Compositions and methods for RNA hybridization applications

ABSTRACT

The invention provides methods and compositions for hybridizing at least one molecule to an RNA target. The invention may, for example, eliminate the use of, or reduce the dependence on formamide in RNA hybridization applications. Compositions for use in the invention include an aqueous composition comprising at least one nucleic acid sequence and at least one polar aprotic solvent in an amount effective to enable hybridization to RNA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a U.S. national stage application of International ApplicationNo. PCT/IB2009/007725, filed Dec. 2. 2009, and also claims the benefitof U.S. Provisional Patent Application No. 61/155,683, filed Feb. 26,2009.

FIELD OF THE INVENTION

The present invention relates to aqueous compositions for use in RNAhybridization applications, for example, for use in in situhybridization (ISH) applications.

In one embodiment, the present invention relates to the field ofmolecular examination of RNA. In particular, the invention relates tothe fields of cytology, histology, and molecular biology. In one aspect,the present invention relates to the energy (e.g., incubation time andheat) required during hybridization between ribonucleic acids, e.g., inin situ hybridization of RNA.

BACKGROUND AND DESCRIPTION

In a basic example of hybridization, nucleic acid probes or primers aredesigned to bind, or “hybridize,” with a target nucleic acid, forexample RNA, in a sample. One type of hybridization application, in situhybridization (ISH), includes hybridization to a target in a specimenwherein the specimen may be in vivo, in situ, or for example, fixed oradhered to a glass slide (i.e., in vitro). Probes may then be used, forexample, to detect genetic abnormalities in a target sequence, providingvaluable information about, e.g., prenatal disorders, cancer, and othergenetic or infectious diseases.

The efficiency and accuracy of nucleic acid hybridization assays mostlydepend on at least one of three major factors: a) denaturation (i.e.,separation of, e.g., two nucleic acid strands) conditions, b)renaturation (i.e., re-annealing of, e.g., two nucleic acid strands)conditions, and c) post-hybridization washing conditions.

In order for the probes or primers to bind to the target nucleic acid inthe sample, complementary strands of nucleic acid must be separated(i.e., denatured). Although RNA is a single-stranded molecule and,therefore, should not require a strand-separation step in order to bindto the primer/probe, RNA molecules can pair with single-stranded DNAmolecules or with other RNA molecules. These associations are stabilizedby hydrogen bonding between bases on opposite strands when bases arepaired in a particular way (A+T/U or G+C) and by hydrophobic bondingamong the stacked bases. In addition, complementary base-pairing andother types of hydrogen bonds can occur between nucleotides in the sameRNA molecule, causing parts of the RNA to fold and pair with itself in adouble helical configuration. Thus, some RNA hybridization applicationsmay optionally include a denaturation step.

Traditional hybridization experiments, such as ISH assays, use hightemperatures (e.g., 95° C. to 100° C.) and/or formamide-containingsolutions to denature doubled stranded nucleic acid. Formamide disruptsbase pairing by displacing loosely and uniformly bound hydrate moleculesand by causing “formamidation” of the Watson-Crick binding sites. Thus,formamide has a destabilizing effect on double stranded nucleic acidsand analogs. However, formamide is a toxic, hazardous material, subjectto strict regulations for use and waste. In addition, the use of highformamide concentrations appears to cause morphological destruction ofcellular, nuclear, and/or chromosomal structure. Heat can also bedestructive to the sample because the phosphodiester bonds of thenucleic acids may be broken at high temperatures, leading to acollection of broken single stranded nucleic acids. In addition, heatcan lead to complications when small volumes are used, since evaporationof aqueous buffers is difficult to control.

Once any double-stranded nucleic acids have been separated, a“renaturation” or “reannealing” step allows the primers or probes tobind to the target nucleic acid in the sample. This step is alsosometimes referred to as the “hybridization” step. The re-annealing stepis by far the most time-consuming aspect of traditional hybridizationapplications. See FIGS. 1 and 2 (presenting examples of traditionalhybridization times for DNA templates). In addition, the presence offormamide in a hybridization buffer can significantly prolong therenaturation time, as compared to aqueous denaturation solutions withoutformamide.

After the probe has annealed to the target nucleic acid in the sample,any unbound and mis-paired probe is removed by a series ofpost-hybridization washes. The specificity of the interaction betweenthe probe and the target is largely determined by stringency of thesepost-hybridization washes. Duplexes containing highly complementarysequences are more resistant to high-stringency conditions than duplexeswith low complementary. Thus, increased stringency conditions can beused to remove non-specific bonds between the probe and the targetnucleic acids. Four variables are typically adjusted to influence thestringency of the post-hybridization washes: (1) temperature (astemperature increases, non-perfect matches between the probe and thetarget sequence will denature, i.e., separate, before more perfectlymatched sequences); (2) salt conditions (as salt concentrationdecreases, non-perfect matches between the probe and the target sequencewill denature, i.e., separate, before more perfectly matched sequences);(3) formamide concentration (as the amount of formamide increases,non-perfect matches between the probe and the target sequence willdenature, i.e., separate, before more perfectly matched sequences); and(4) time (as the wash time increases, non-perfect matches between theprobe and the target sequence will denature, i.e., separate, before moreperfectly matched sequences).

The present invention provides several potential advantages over priorart methods and compositions for RNA hybridization applications. Theseadvantages include faster hybridization times, lower hybridizationtemperatures, and less toxic hybridization solvents.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide compositions whichresult in at least one of the following advantages: highly sensitive,technically easy, fast, flexible, and reliable RNA hybridizationprocedures that are easy to automate, preserve sample morphology,provide safer reagents, reduce background, and reduce evaporation ofreagent. Yet another object of the invention is to provide a compositionwith a low probe concentration. It is another object of the invention toreduce and/or remove the need for blocking of unspecific binding. Thecompositions and methods of the invention may also permit the use ofheterogeneous probes without the need to block, remove, or otherwisedisable the binding of, e.g., repetitive sequences in a biologicalsample or loop formation of RNA.

The compositions and methods of the invention are applicable to any RNAhybridization technique. The compositions and methods of the inventionare also applicable to any molecules that hybridize or bind to RNA usingbase pairing, such as, for example, single-stranded DNA, RNA, PNA, LNA,and synthetic and natural analogs thereof.

In one embodiment, the nucleic acid hybridization method andcompositions of the present invention are useful for the in vitro, invivo, or in situ analysis of cellular RNA. Further, the methods andcompositions are useful for detection of RNA from infectious agents aswell as changes in levels of expression of RNA.

For example, the method and compositions of the invention may be usedfor the in vitro, in vivo, or in situ analysis of messenger RNA (mRNA),viral RNA, small interfering RNA (siRNA), small nuclear RNA (snRNA),non-coding RNA (ncRNA, e.g., tRNA and rRNA), transfer messenger RNA(tmRNA), micro RNA (miRNA), piwi-interacting RNA (piRNA), long noncodingRNA, small nucleolar RNA (snoRNA), antisense RNA, double-stranded RNA(dsRNA), and heterogeneous nuclear RNA (hnRNA).

The nucleic acid hybridization method and compositions of the presentinvention are useful for in vitro, in vivo, or in situ analysis of RNAusing techniques such as northern blot, flow cytometry, autoradiography,fluorescence microscopy, chemiluminescence, immunohistochemistry,virtual karyotype, gene assay, microarray, gene expression profiling,Gene ID, Tiling array, gel electrophoresis, capillary electrophoresis,and in situ hybridizations such as FISH, SISH, CISH.

The methods and compositions of the invention may be used on in situ, invitro, and in vivo samples such as bone marrow smears, blood smears,paraffin embedded tissue preparations, enzymatically dissociated tissuesamples, bone marrow, amniocytes, cytospin preparations, imprints, etc.

In one embodiment, the invention provides methods and compositions forhybridizing at least one molecule to a target RNA. In one embodiment,the compositions and methods of the invention lower the energy necessaryfor hybridization applications. The lower energy barrier may reduce thetime and/or temperature necessary for hybridization applications. Forexample, the invention may allow for performing the hybridization stepat lower temperatures or may allow for a rapid hybridization step athigher temperatures. In addition, the hybridization step may beperformed at room temperature. Thus, the methods and compositions of theinvention enable one to tailor the time required for hybridizationapplications by varying the temperature of the reaction to a muchgreater degree than is available using prior art methods, overcoming amajor time-consuming step in hybridization assays.

The invention may also eliminate the use of, or reduce the dependence onformamide. For example, the methods and compositions of the inventionmay lower the energy barrier to hybridization without the use offormamide.

One aspect of the invention is a composition or solution for use in RNAhybridization applications. Compositions for use in the inventioninclude an aqueous composition comprising at least one nucleic acidsequence and at least one polar aprotic solvent in an amount effectiveto enable hybridization to RNA. One way to test for whether the amountof polar aprotic solvent is effective to enable hybridization to RNA isto determine whether the polar aprotic solvent, when used in thehybridization methods and compositions described herein, such as example1, yield a detectable signal.

Non-limiting examples of effective amounts of polar aprotic solventsinclude, e.g., about 1% to about 95% (v/v). In some embodiments, theconcentration of polar aprotic solvent is 5% to 60% (v/v). In otherembodiments, the concentration of polar aprotic solvent is 10% to 60%(v/v). In still other embodiments, the concentration of polar aproticsolvent is 30% to 50% (v/v). Concentrations of 1% to 5%, 5% to 10%, 10%,10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or 50% to 60% (v/v) arealso suitable. In some embodiments, the polar aprotic solvent will bepresent at a concentration of 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, or 5%(v/v). In other embodiments, the polar aprotic solvent will be presentat a concentration of 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%,11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%,17.5%, 18%, 18.5%, 19%, 19.5%, or 20% (v/v).

According to another aspect of the present invention the aqueouscomposition comprising a polar aprotic solvent has reduced toxicity. Forexample, a less-toxic composition than traditional solutions used inhybridization applications may comprise a composition with the provisothat the composition does not contain formamide, or with the provisothat the composition contains less than 10%, or less than 5%, or lessthan 2%, or less than 1%, or less than 0.5%, or less than 0.1%, or lessthan 0.05%, or less than 0.01% formamide. A less-toxic composition mayalso comprise a composition with the proviso that the composition doesnot contain dimethyl sulfoxide (DMSO), or with the proviso that thecomposition contains less than 10%, 5%, 2%, or less than 1%, or lessthan 0.5%, or less than 0.1%, or less than 0.05%, or less than 0.01%DMSO.

In one aspect of the invention, suitable polar aprotic solvents for usein the invention may be selected based on their Hansen SolubilityParameters. For example, suitable polar aprotic solvents may have adispersion solubility parameter between 17.7 to 22.0 MPa^(1/2), a polarsolubility parameter between 13 to 23 MPa^(1/2), and a hydrogen bondingsolubility parameter between 3 to 13 MPa^(1/2).

According to one aspect of the present invention, suitable polar aproticsolvents for use in the invention are cyclic compounds. A cycliccompound has a cyclic base structure. Examples include the cycliccompounds disclosed herein. In other embodiments, the polar aproticsolvent may be chosen from Formulas 1-4 below:

where X is O and R1 is alkyldiyl.

According to another aspect of the invention, suitable polar aproticsolvents for use in the invention may be chosen from Formula 5 below:

where X is optional and if present, is chosen from O or S;where Z is optional and if present, is chosen from O or S;where A and B independently are O or N or S or part of the alkyldiyl ora primary amine;where R is alkyldiyl; andwhere Y is O or S or C.

Examples of suitable polar aprotic solvents according to Formula 5 areprovided in Formulas 6-9 below:

According to yet another aspect of the invention the polar aproticsolvent has lactone, sulfone, nitrile, sulfite, or carbonatefunctionality. Such compounds are distinguished by their relatively highdielectric constants, high dipole moments, and solubility in water.

According to another aspect of the invention the polar aprotic solventhaving lactone functionality is γ-butyrolactone (GBL), the polar aproticsolvent having sulfone functionality is sulfolane (SL), the polaraprotic solvent having nitrile functionality is acetonitrile (AN), thepolar aprotic solvent having sulfite functionality is glycolsulfite/ethylene sulfite (GS), and the polar aprotic solvent havingcarbonate functionality is ethylene carbonate (EC), propylene carbonate(PC), or ethylene thiocarbonate (ETC). In yet another aspect of theinvention, the compositions and methods of the invention comprise apolar aprotic solvent, with the proviso that the polar aprotic solventis not acetonitrile (AN) or sulfolane (SL).

In one embodiment, the at least one nucleic acid sequence is a PNAsequence. In another embodiment, the at least one nucleic acid sequenceis an LNA sequence.

According to yet another aspect, the invention discloses a hybridizationmethod comprising:

-   -   providing an RNA sequence,    -   providing a second nucleic acid sequence,    -   providing an aqueous composition comprising at least one polar        aprotic solvent in an amount effective to enable hybridization        to RNA, and    -   combining the RNA and the second nucleic acid sequence and the        aqueous composition for at least a time period sufficient to        hybridize the RNA and the second nucleic acid sequence.

According to yet another aspect, the invention discloses a hybridizationmethod comprising:

-   -   providing an RNA sequence, and    -   applying to said RNA sequence an aqueous composition comprising        a second nucleic acid sequence and at least one polar aprotic        solvent in an amount effective to enable hybridization to RNA        for at least a time period sufficient to hybridize the RNA and        the second nucleic acid sequences.

In one embodiment, the RNA is in a biological sample. In anotherembodiment, the biological sample is a cytology or histology sample. Inone embodiment, the second nucleic acid sequence is a PNA sequence, anLNA sequence, or a DNA sequence.

In one embodiment, the step of hybridizing the RNA sequence to thesecond nucleic acid sequence occurs in less than 8 hours, such as, forexample, less than 6 hours, less than 5 hours, less than 4 hours, lessthan 3 hours, less than 2 hours, or less than 1 hour. According toanother aspect the present invention relates to a method wherein thehybridization step takes less than 1 hour. In other embodiments, thehybridization step takes less than 30 minutes. In still otherembodiments, the hybridization step takes less than 15 minutes. In otherembodiments, the hybridization step takes less than 5 minutes.

In one embodiment, a sufficient amount of energy to hybridize the RNAand the second nucleic acids is provided.

According to yet another aspect of the present invention, thehybridization energy is provided by heating the aqueous composition andnucleic acid sequence. Thus, the step of hybridizing may include thesteps of heating and cooling the aqueous composition and nucleic acidsequences.

A further aspect of the invention comprises a method wherein the step ofproviding a sufficient amount of energy to hybridize the nucleic acidsinvolves a heating step performed by the use of microwaves, hot baths,hot plates, heat wire, peltier element, induction heating, or heatlamps.

According to another aspect of the invention, the method furtherincludes a denaturation step. In one embodiment, the nucleic acidsequences are denatured separately. For example, the specimen may bedenatured with a solution without probe and thereafter hybridized withprobe. In another embodiment, the nucleic acid sequences are denaturedtogether.

According to a further aspect, the invention relates to the use of acomposition comprising between 1 and 95% (v/v) of at least one polaraprotic solvent in an RNA hybridization application.

According to yet another aspect, the invention relates to the use of acomposition comprising an aqueous composition as described in thisinvention for use in an RNA hybridization application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a typical time-course for single locus detection of a DNAtarget with primary labeled FISH probes on formaldehyde fixed paraffinembedded tissue sections (histological specimens). The bars represent ahybridization assay performed using a traditional solution (top) and atypical time-course for a hybridization assay performed using acomposition of the invention (bottom). The first bar on the left in eachtime-course represents the deparaffination step; the second barrepresents the heat-pretreatment step; the third bar represents thedigestion step; the fourth bar represents the denaturation andhybridization steps; the fifth bar represents the stringency wash step;and the sixth bar represents the mounting step.

FIG. 2 depicts a typical time-course for single locus detection of a DNAtarget with primary labeled FISH probes on cytological specimens. Thebars represent a hybridization assay performed using a traditionalsolution (top) and a typical time-course for a hybridization assayperformed using a composition of the invention (bottom). The first baron the left in each time-course represents the fixation step; the secondbar represents the denaturation and hybridization steps; the third barrepresents the stringency wash step; and the fourth bar represents themounting step.

DETAILED DESCRIPTION A. Definitions

In the context of the present invention the following terms are to beunderstood as follows:

“Biological sample” is to be understood as any in vivo, in vitro, or insitu sample of one or more cells or cell fragments. This can, forexample, be a unicellular or multicellular organism, tissue section,cytological sample, chromosome spread, purified nucleic acid sequences,artificially made nucleic acid sequences made by, e.g., a biologic basedsystem or by chemical synthesis, microarray, or other form of nucleicacid chip. In one embodiment, a sample is a mammalian sample, such as,e.g., a human, murine, rat, feline, or canine sample.

“Nucleic acid,” “nucleic acid chain,” and “nucleic acid sequence” meananything that binds or hybridizes using base pairing including,oligomers or polymers having a backbone formed from naturally occurringnucleotides and/or nucleic acid analogs comprising nonstandardnucleobases and/or nonstandard backbones (e.g., a peptide nucleic acid(PNA) or locked nucleic acid (LNA)), or any derivatized form of anucleic acid.

As used herein, the term “peptide nucleic acid” or “PNA” means asynthetic polymer having a polyamide backbone with pendant nucleobases(naturally occurring and modified), including, but not limited to, anyof the oligomer or polymer segments referred to or claimed as peptidenucleic acids in, e.g., U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049,5,714,331, 5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461,5,837,459, 5,891,625, 5,972,610, 5,986,053, 6,107,470 6,201,103,6,228,982 and 6,357,163, WO96/04000, all of which are hereinincorporated by reference, or any of the references cited therein. Thependant nucleobase, such as, e.g., a purine or pyrimidine base on PNAmay be connected to the backbone via a linker such as, e.g., one of thelinkers taught in PCT/US02/30573 or any of the references cited therein.In one embodiment, the PNA has an N-(2-aminoethyl)-glycine) backbone.PNAs may be synthesized (and optionally labeled) as taught inPCT/US02/30573 or any of the references cited therein. PNAs hybridizetightly, and with high sequence specificity, with DNA and RNA, becausethe PNA backbone is uncharged. Thus, short PNA probes may exhibitcomparable specificity to longer DNA or RNA probes. PNA probes may alsoshow greater specificity in binding to complementary DNA or RNA.

As used herein, the term “locked nucleic acid” or “LNA” means anoligomer or polymer comprising at least one or more LNA subunits. Asused herein, the term “LNA subunit” means a ribonucleotide containing amethylene bridge that connects the 2′-oxygen of the ribose with the4′-carbon. See generally, Kurreck, Eur. J. Biochem., 270:1628-44 (2003).

Examples of nucleic acids and nucleic acid analogs also include polymersof nucleotide monomers, including double and single strandeddeoxyribonucleotides (DNA), ribonucleotides (RNA), α-anomeric formsthereof, synthetic and natural analogs thereof, and the like. Thenucleic acid chain may be composed entirely of deoxyribonucleotides,ribonucleotides, peptide nucleic acids (PNA), locked nucleic acids(LNA), synthetic or natural analogs thereof, or mixtures thereof. DNA,RNA, or other nucleic acids as defined herein can be used in the methodand compositions of the invention.

“Polar aprotic solvent” refers to an organic solvent having a dipolemoment of about 2 debye units or more, a water solubility of at leastabout 5% (volume) at or near ambient temperature, i.e., about 20° C.,and which does not undergo significant hydrogen exchange atapproximately neutral pH, i.e., in the range of 5 to 9, or in the range6 to 8. Polar aprotic solvents include those defined according to theHansen Solubility Parameters discussed below.

“Alkyldiyl” refers to a saturated or unsaturated, branched, straightchain or cyclic hydrocarbon radical having two monovalent radicalcenters derived by the removal of one hydrogen atom from each of twodifferent carbon atoms of a parent alkane, alkene, or alkyne.

“Aqueous solution” is to be understood as a solution containing water,even small amounts of water. For example, a solution containing 1% wateris to be understood as an aqueous solution.

“Hybridization application,” “hybridization assay,” “hybridizationexperiment,” “hybridization procedure,” “hybridization technique,”“hybridization method,” etc. are to be understood as referring to anyprocess that involves hybridization to RNA. Unless otherwise specified,the terms “hybridization” and “hybridization step” are to be understoodas referring to the re-annealing step of the RNA hybridization procedureas well as the optional denaturation step (if present).

“Hybridization composition” refers to an aqueous solution of theinvention for performing a hybridization procedure, for example, to binda probe to a nucleic acid sequence. Hybridization compositions maycomprise, e.g., at least one polar aprotic solvent, at least one nucleicacid sequence, and a hybridization solution. Hybridization compositionsdo not comprise enzymes or other components, such as deoxynucleosidetriphosphates (dNTPs), for amplifying nucleic acids in a biologicalsample.

“Hybridization solution” refers to an aqueous solution for use in ahybridization composition of the invention. Hybridization solutions arediscussed in detail below and may comprise, e.g., buffering agents,accelerating agents, chelating agents, salts, detergents, and blockingagents.

“Hansen Solubility Parameters” and “HSP” refer to the following cohesionenergy (solubility) parameters: (1) the dispersion solubility parameter(δ_(D), “D parameter”), which measures nonpolar interactions derivedfrom atomic forces; (2) the polar solubility parameter (δ_(P), “Pparameter”), which measures permanent dipole-permanent dipoleinteractions; and (3) the hydrogen bonding solubility parameter (δ_(H),“H parameter”), which measures electron exchange. The Hansen SolubilityParameters are further defined below.

“Repetitive Sequences” is to be understood as referring to the rapidlyreannealing (approximately 25%) and/or intermediately reannealing(approximately 30%) components of mammalian genomes. The rapidlyreannealing components contain small (a few nucleotides long) highlyrepetitive sequences usually found in tandem (e.g., satellite DNA),while the intermediately reannealing components contain interspersedrepetitive DNA. Interspersed repeated sequences are classified as eitherSINEs (short interspersed repeat sequences) or LINEs (long interspersedrepeated sequences), both of which are classified as retrotransposons inprimates. SINEs and LINEs include, but are not limited to, Alu-repeats,Kpn-repeats, di-nucleotide repeats, tri-nucleotide repeats,tetra-nucleotide repeats, penta-nucleotide repeats and hexa-nucleotiderepeats. Alu repeats make up the majority of human SINEs and arecharacterized by a consensus sequence of approximately 280 to 300 bpthat consist of two similar sequences arranged as a head to tail dimer.In addition to SINEs and LINES, repeat sequences also exist inchromosome telomeres at the termini of chromosomes and chromosomecentromeres, which contain distinct repeat sequences that exist only inthe central region of a chromosome. However, unlike SINEs and LINEs,which are dispersed randomly throughout the entire genome, telomere andcentromere repeat sequences are localized within a certain region of thechromosome.

“Non-toxic” and “reduced toxicity” are defined with respect to thetoxicity labeling of formamide according to “Directive 1999/45/EC of theEuropean Parliament and of the Council of 31 May 1999 concerning theapproximation of the laws, regulations and administrative provisions ofthe Member States relating to the classification, packaging, andlabelling of dangerous preparations”(ecb.jrc.it/legislation/1999L0045EC.pdf) (“Directive”). According to theDirective, toxicity is defined using the following classification order:T+“very toxic”; T “toxic”, C “corrosive”, Xn “harmful”, .Xi “irritant.”Risk Phrases (“R phrases”) describe the risks of the classifiedtoxicity. Formamide is listed as T (toxic) and R61 (may cause harm tothe unborn child). All of the following chemicals are classified as lesstoxic than formamide: acetonitrile (Xn, R11, R20, R21, R22, R36);sulfolane (Xn, R22); γ-butyrolactone (Xn, R22, R32); and ethylenecarbonate (Xi, R36, R37, R38). At the time of filing this application,ethylene trithiocarbonate and glycol sulfite are not presently labeled.

“Stringent” and “stringency” in the context of post-hybridization washesare to be understood as referring to conditions for reducingnon-complementary base-pairing. In general, a signal to noise ratio of2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates specific hybridization withlittle non-complementary base-pairing. The specificity of theinteraction between the probe and the target is largely determined bystringency of these post-hybridization washes. Duplexes containinghighly complementary sequences are more resistant to high-stringencyconditions than duplexes with low complementary. Thus, increasedstringency conditions can be used to remove non-specific bonds betweenthe probe and the target nucleic acids. Four variables are typicallyadjusted to influence the stringency of the post-hybridization washes:(1) temperature (as temperature increases, non-perfect matches betweenthe probe and the target sequence will denature, i.e., separate, beforemore perfectly matched sequences); (2) salt conditions (as saltconcentration decreases, non-perfect matches between the probe and thetarget sequence will denature, i.e., separate, before more perfectlymatched sequences); (3) formamide concentration (as the amount offormamide increases, non-perfect matches between the probe and thetarget sequence will denature, i.e., separate, before more perfectlymatched sequences); and (4) time (as the wash time increases,non-perfect matches between the probe and the target sequence willdenature, i.e., separate, before more perfectly matched sequences).Other factors such as pH, rate of agitation, and number of washes willalso influence the stringency of the wash step.

As used herein, the terms “room temperature” and “RT” refer to about 20°C. to about 25° C., unless otherwise stated.

B. Solvent Selection

Suitable polar aprotic solvents for use in the invention may be selectedbased on their Hansen Solubility Parameters. Methods for experimentallydetermining and/or calculating HSP for a solvent are known in the art,and HSP have been reported for over 1200 chemicals.

For example, the D parameter may be calculated with reasonable accuracybased on refractive index, or may be derived from charts by comparisonwith known solvents of similar size, shape, and composition afterestablishing a critical temperature and molar volume. The P parametermay be estimated from known dipole moments (see, e.g., McClellan A. L.,Tables of Experimental Dipole Moments (W.H. Freeman 1963)) usingEquation 1:δ_(P)=37.4(Dipole Moment)/V ^(1/2)  Equation 1where V is the molar volume. There are no equations for calculating theH parameter. Instead, the H parameter is usually determined based ongroup contributions.

HSP characterizations are conveniently visualized using a sphericalrepresentation, with the HSP of an experimentally-determined suitablereference solvent at the center of the sphere. The radius of the sphere(R) indicates the maximum tolerable variation from the HSP of thereference solvent that still allows for a “good” interaction to takeplace. Good solvents are within the sphere and bad ones are outside. Thedistance, R_(a), between two solvents based on their respective HSPvalues can be determined using Equation 2:(R _(a))²=4(δ_(D1)−δ_(D2))²+(δ_(P1)−δ_(P2))²(δ_(H1)−δ_(H2))²  Equation 2where subscript 1 indicates the reference sample, subscript 2 indicatesthe test chemical, and all values are in MPa^(1/2). Good solubilityrequires that R_(a) be less than the experimentally-determined radius ofthe solubility sphere R_(o). The relative energy difference between twosolvents, i.e., RED number, can be calculated by taking the ratio ofR_(a) to R_(o), as shown in Equation 3.RED=R _(a) /R _(o)  Equation 3RED numbers less than 1.0 indicate high affinity; RED numbers equal orclose to 1.0 indicate boundary conditions; and progressively higher REDnumbers indicate progressively lower affinities.

In some embodiments, the D parameters of the polar aprotic solvents ofthe invention are between 17.7 to 22.0 MPa^(1/2). Such relatively high Dparameters are generally associated with solvents having cyclicstructures and/or structures with sulfur or halogens. Linear compoundsare not likely to be among the most suitable polar aprotic solvents foruse in the invention, but may be considered if their P and H parametersare within the ranges discussed below. Since the D parameter ismultiplied by 4 in Equation 2, the limits are one-half of R_(o). Inaddition, it should be noted that D values of around 21 or higher areoften characteristic of a solid.

In some embodiments, the P parameters of the polar aprotic solvents ofthe invention are between 13 to 23 MPa^(1/2). Such exceptionally high Pparameters are generally associated with solvents having a high dipolemoment and presumably also a relatively low molecular volume. Forexample, for V near 60 cc/mole, the dipole moment should be between 4.5and 3.1. For V near 90 cc/mole, the dipole moment should be between 5.6and 3.9.

In some embodiments, the H parameters of the polar aprotic solvents ofthe invention are between 3 to 13 MPa^(1/2). Generally, polar aproticsolvents having an alcohol group are not useful in the compositions andmethods of the invention, since the H parameters of such solvents wouldbe too high.

The molar volume of the polar aprotic solvent may also be relevant,since it enters into the evaluation of all three Hansen SolubilityParameters. As molar volume gets smaller, liquids tend to evaporaterapidly. As molar volume gets larger, liquids tend to enter the solidregion in the range of D and P parameters recited above. Thus, the polaraprotic solvents of the invention are rather close to the liquid/solidboundary in HSP space.

In some embodiments, the polar aprotic solvents of the invention havelactone, sulfone, nitrile, sulfite, and/or carbonate functionality. Suchcompounds are distinguished by their relatively high dielectricconstants, high dipole moments, and solubility in water. An exemplarypolar aprotic solvent with lactone functionality is γ-butyrolactone(GBL), an exemplary polar aprotic solvent with sulfone functionality issulfolane (SL; tetramethylene sulfide-dioxide), an exemplary polaraprotic solvent with nitrile functionality is acetonitrile (AN), anexemplary polar aprotic solvent with sulfite functionality is glycolsulfite/ethylene sulfite (GS), and an exemplary polar aprotic solventswith carbonate functionality are ethylene carbonate (EC), propylenecarbonate (PC), or ethylene trithiocarbonate (ETC). The structures ofthese exemplary solvents are provided below and their Hansen SolubilityParameters, RED numbers, and molar volumes are given in Table 1.

TABLE 1 Molar Volume D P H RED (cm³/mole) Correlation 19.57 19.11 7.71 —— (R₀ = 3.9) GBL 19.0 16.6 7.4 0.712 76.5 PC 20.0 18.0 4.1 0.993 85.2 SL20.3 18.2 10.9 0.929 95.7 EC 19.4 21.7 5.1 0.946 66.0 ETC n/a n/a n/an/a n/a GS 20.0 15.9 5.1 n/a 75.1 n/a = not available.

Other suitable polar aprotic solvents that may be used in the inventionare cyclic compounds such as, e.g., ε-caprolactone. In addition,substituted pyrrolidinones and related structures with nitrogen in a 5-or 6-membered ring, and cyclic structures with two nitrile groups, orone bromine and one nitrile group, may also be suitable for use in theinvention. For example, N-methylpyrrolidinone (shown below) may be asuitable polar aprotic solvent for use in the methods and compositionsof the invention.

Other suitable polar aprotic solvents may contain a ring urethane group(NHCOO—). However, not all such compounds are suitable, since1,3-dimethyl-2-imidazolidinone produces no signals when used in thehybridization compositions of the invention. One of skill in the art mayscreen for compounds useful in the compositions and methods of theinvention as described herein. Exemplary chemicals that may be suitablefor use in the invention are set forth in Tables 2 and 3 below.

TABLE 2 Solvent D P H Acetanilide 20.6 13.3 12.4 N-Acetyl Pyrrolidone17.8 13.1 8.3 4-Amino Pyridine 20.4 16.1 12.9 Benzamide 21.2 14.7 11.2Benzimidazole 20.6 14.9 11.0 1,2,3-Benzotriazole 18.7 15.6 12.4Butadienedioxide 18.3 14.4 6.2 2,3-Butylene Carbonate 18.0 16.8 3.1Caprolactone (Epsilon) 19.7 15.0 7.4 Chloro Maleic Anhydride 20.4 17.311.5 2-Chlorocyclohexanone 18.5 13.0 5.1 Chloronitromethane 17.4 13.55.5 Citraconic Anhydride 19.2 17.0 11.2 Crotonlactone 19.0 19.8 9.6Cyclopropylnitrile 18.6 16.2 5.7 Dimethyl Sulfate 17.7 17.0 9.7 DimethylSulfone 19.0 19.4 12.3 Dimethyl Sulfoxide 18.4 16.4 10.21,2-Dinitrobenzene 20.6 22.7 5.4 2,4-Dinitrotoluene 20.0 13.1 4.9Dipheynyl Sulfone 21.1 14.4 3.4 1,2-Dinitrobenzene 20.6 22.7 5.42,4-Dinitrotoluene 20.0 13.1 4.9 Epsilon-Caprolactam 19.4 13.8 3.9Ethanesulfonylchloride 17.7 14.9 6.8 Furfural 18.6 14.9 5.12-Furonitrile 18.4 15.0 8.2 Isoxazole 18.8 13.4 11.2 Maleic Anhydride20.2 18.1 12.6 Malononitrile 17.7 18.4 6.7 4-Methoxy Benzonitrile 19.416.7 5.4 1-Methoxy-2-Nitrobenzene 19.6 16.3 5.5 1-Methyl Imidazole 19.715.6 11.2 3-Methyl Isoxazole 19.4 14.8 11.8 N-Methyl Morpholine-N- 19.016.1 10.2 Oxide Methyl Phenyl Sulfone 20.0 16.9 7.8 Methyl Sulfolane19.4 17.4 5.3 Methyl-4-Toluenesulfonate 19.6 15.3 3.8 3-Nitroaniline21.2 18.7 10.3 2-Nitrothiophene 19.7 16.2 8.2 9,10-Phenanthrenequinone20.3 17.1 4.8 Phthalic Anhydride 20.6 20.1 10.1 1,3-Propane Sultone 18.416.0 9.0 beta-Propiolactone 19.7 18.2 10.3 2-Pyrrolidone 19.4 17.4 11.3Saccharin 21.0 13.9 8.8 Succinonitrile 17.9 16.2 7.9 Sulfanilamide 20.019.5 10.7 Sulfolane 20.3 18.2 10.9 2,2,6,6- 19.5 14.0 6.3Tetrachlorocyclohexanone Thiazole 20.5 18.8 10.8 3,3,3-Trichloro Propene17.7 15.5 3.4 1,1,2-Trichloro Propene 17.7 15.7 3.4 1,2,3-TrichloroPropene 17.8 15.7 3.4

Table 2 sets forth an exemplary list of potential chemicals for use inthe compositions and methods of the invention based on their HansenSolubility Parameters. Other compounds, may of course, also meet theserequirements such as, for example, those set forth in Table 3.

TABLE 3 Chemical (dipole moment) RED Melting Point ° C. Chloroethylenecarbonate (4.02) 0.92 — 2-Oxazolidinone (5.07) 0.48 86-89 2-Imidazole1.49 90-91 1,5-Dimethyl Tetrazole (5.3) ~1.5 70-72 N-Ethyl Tetrazole(5.46) ~1.5 Trimethylene sulfide-dioxide (4.49) — — Trimethylene sulfite(3.63) — — 1,3-Dimethyl-5-Tetrazole (4.02) — — Pyridazine (3.97) 1.16 −82-Thiouracil (4.21) — — N-Methyl Imidazole (6.2) 1.28 —1-Nitroso-2-pyrolidinone ~1.37 — Ethyl Ethyl Phosphinate (3.51) — —5-cyano-2-Thiouracil (5.19) — — 4H-Pyran-4-thione (4.08) 1.35 32-344H-Pyran-4-one = gamma pyrone (4.08) 1.49 Boiling Point (BP) 802-Nitrofuran (4.41) 1.14 29 Methyl alpha Bromo Tetronate (6.24) — —Tetrahydrothiapyran oxide (4.19) 1.75 60-64 Picolinonitrile(2-cyanopyridine) (5.23) 0.40 26-28 (BP 212-215) Nitrobenzimidazole(6.0) 0.52 207-209 Isatin (5.76) — 193-195 N-phenyl sydnone (6.55) — —Glycol sulfate (Ethylene glycol) — 99° C. Note: not soluble at 40%

Some of the chemicals listed in Tables 2 and 3 have been used inhybridization and/or PCR applications in the prior art (e.g., dimethylsulfoxide (DMSO) has been used in hybridization and PCR applications,and sulfolane (SL), acetonitrile (AN), 2-pyrrolidone, ε-caprolactam, andethylene glycol have been used in PCR applications). Thus, in someembodiments, the polar aprotic solvent is not DMSO, sulfolane,acetonitrile, 2-pyrrolidone, ε-caprolactam, or ethylene glycol. However,most polar aprotic solvents have not been used in prior arthybridization applications. Moreover, even when such compounds wereused, the prior art did not recognize that they may be advantageouslyused to decrease hybridization times and/or temperatures in RNAhybridizations, as disclosed in this application.

In addition, not all of the chemicals listed in Tables 2 and 3 aresuitable for use in the compositions and methods of the invention. Forexample, although DMSO is listed in Table 2 because its HansenSolubility Parameters (HSPs) fall within the ranges recited above, DMSOlikely does not function to decrease hybridization times and/ortemperatures in the compositions and methods of the invention. However,it is well within the skill of the ordinary artisan to screen forsuitable compounds using the guidance provided herein including testinga compound in one of the examples provided. For example, in someembodiments, suitable polar aprotic solvents will have HSPs within theranges recited above and a structure shown in Formulas 1-9 above.

C. Compositions, Buffers, and Solutions

(1) Hybridization Solutions

Traditional hybridization solutions for use in RNA applications areknown in the art. Such solutions may comprise, for example, bufferingagents, accelerating agents, chelating agents, salts, detergents, andblocking agents.

For example, the buffering agents may include SSC, HEPES, SSPE, PIPES,TMAC, TRIS, SET, citric acid, a phosphate buffer, such as, e.g.,potassium phosphate or sodium pyrrophosphate, etc. The buffering agentsmay be present at concentrations from 0.01× to 50×, such as, forexample, 0.01×, 0.1×, 0.5×, 1×, 2×, 5×, 10×, 15×, 20×, 25×, 30×, 35×,40×, 45×, or 50×. Typically, the buffering agents are present atconcentrations from 0.1× to 10×.

The accelerating agents may include polymers such as FICOLL, PVP,heparin, dextran sulfate, proteins such as BSA, glycols such as ethyleneglycol, glycerol, 1,3 propanediol, propylene glycol, or diethyleneglycol, combinations thereof such as Denhardt's solution and BLOTTO, andorganic solvents such as formamide, dimethylformamide, DMSO, etc. Theaccelerating agent may be present at concentrations from 1% to 80% or0.1× to 10×, such as, for example, 0.1% (or 0.1×), 0.2% (or 0.2×), 0.5%(or 0.5×), 1% (or 1×), 2% (or 2×), 5% (or 5×), 10% (or 10×), 15% (or15×), 20% (or 20×), 25% (or 25×), 30% (or 30×), 40% (or 40×), 50% (or50×), 60% (or 60×), 70% (or 70×), or 80% (or 80×). Typically, formamideis present at concentrations from 25% to 75%, such as 25%, 30%, 40%,50%, 60%, 70%, or 75%, while DMSO, dextran sulfate, and glycol arepresent at concentrations from 5% to 10%, such as 5%, 6%, 7%, 8%, 9%, or10%.

The chelating agents may include EDTA, EGTA, etc. The chelating agentsmay be present at concentrations from 0.1 mM to 10 mM, such as 0.1 mM,0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or10 mM. Typically, the chelating agents are present at concentrationsfrom 0.5 mM to 5 mM, such as 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM,3.5 mM, 4 mM, 4.5 mM, or 5 mM.

The salts may include sodium chloride, sodium phosphate, magnesiumphosphate, etc. The salts may be present at concentrations from 1 mM to750 mM, such as 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM,200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, or 750 mM. Typically,the salts are present at concentrations from 10 mM to 500 mM, such as 10mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, or 500mM.

The detergents may include Tween, SDS, Triton, CHAPS, deoxycholic acid,etc. The detergent may be present at concentrations from 0.001% to 10%,such as, for example, 0.0001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10%. Typically, the detergents are present at concentrations from0.01% to 1%, such as 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%.

The nucleic acid blocking agents may include, yeast tRNA, homopolymerDNA, denatured salmon sperm DNA, herring sperm DNA, total human DNA,COT1 DNA, etc. The blocking nucleic acids may be present atconcentrations of 0.05 mg/mL to 100 mg/mL.

A great variation exists in the literature regarding traditionalhybridization solutions. For example, a traditional hybridizationsolution may comprise 5× or 6×SSC, 0.01 M EDTA, 5×Denhardt's solution,0.5% SDS, and 100 mg/mL sheared, denatured salmon sperm DNA. Anothertraditional hybridization solution may comprise 50 mM HEPES, 0.5 M NaCl,and 0.2 mM EDTA. Yet another traditional hybridization solution maycomprise 2×SSC, 10% dextran sulfate, 50% formamide, and e.g., 0.3 mg/mLsalmon sperm DNA or 0.1 mg/mL COT1 DNA. A typical hybridization solutionfor FISH on biological specimens for RNA detection may comprise, e.g.,2×SSC, 10% dextran sulfate, 2 mM vanadyl-ribonucleoside complex, 50%formamide, 0.02% RNAse-free BSA, and 1 mg/mL E. coli tRNA. Other typicalhybridization solutions for DNA detection may comprise 40% formamide,10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, Alu-PNA(blocking PNA) or COT-1 DNA, and in some cases 0.1 μg/μL total human DNA(THD).

The compositions of the invention may comprise a hybridization solutioncomprising any of the components of traditional hybridization solutionsrecited above in combination with at least one polar aprotic solvent.The traditional components may be present at the same concentrations asused in traditional hybridization solutions, or may be present at higheror lower concentrations, or may be omitted completely.

For example, if the compositions of the invention comprise salts such asNaCl and/or phosphate buffer, the salts may be present at concentrationsof 0-1200 mM NaCl and/or 0-200 mM phosphate buffer. In some embodiments,the concentrations of salts may be, for example, 300 mM NaCl and 5 mMphosphate buffer, or 600 mM NaCl and 10 mM phosphate buffer.

If the compositions of the invention comprise accelerating agents suchas dextran sulfate, glycol, or DMSO, the dextran sulfate may be presentat concentrations of from 5% to 40%, the glycol may be present atconcentrations of from 0.1% to 10%, and the DMSO may be from 0.1% to10%. In some embodiments, the concentration of dextran sulfate may be10% or 20% and the concentration of ethylene glycol, 1,3 propanediol, orglycerol may be 1% to 10%. In some embodiments, the concentration ofDMSO may be 1%. In some embodiments, the aqueous composition does notcomprise DMSO as an accelerating agent. In some embodiments, the aqueouscomposition does not comprise formamide as an accelerating agent, orcomprises formamide with the proviso that the composition contains lessthan 10%, or less than 5%, or less than 2%, or less than 1%, or lessthan 0.5%, or less than 0.1%, or less than 0.05%, or less than 0.01%.

If the compositions of the invention comprise citric acid, theconcentrations may range from 1 mM to 50 mM and the pH may range from5.0 to 8.0. In some embodiments the concentration of citric acid may be10 mM and the pH may be 6.2.

The compositions of the invention may comprise agents that reducenon-specific binding to, for example, the cell membrane, such as salmonsperm DNA or small amounts of total human DNA or, for example, they maycomprise blocking agents to block binding of, e.g., repeat sequences inthe target, such as larger amounts of total human DNA or repeat-enrichedDNA, or specific blocking agents such as PNA or LNA fragments andsequences. These agents may be present at concentrations of from0.01-100 μg/μL or 0.01-100 μM. For example, in some embodiments, theseagents will be 0.1 μg/μL total human DNA, or 0.1 μg/μL non-human DNA,such as herring sperm, salmon sperm, or calf thymus DNA, or 5 μMblocking PNA.

One aspect of the invention is a composition or solution for use in RNAhybridization applications. Compositions for use in the inventioninclude an aqueous composition comprising a nucleic acid sequence and atleast one polar aprotic solvent in an amount effective to enablehybridization to RNA. One way to test for whether the amount of polaraprotic solvent is effective to enable hybridization to RNA is todetermine whether the polar aprotic solvent, when used in thehybridization methods and compositions described herein, such as example1, yield a detectable signal.

Non-limiting examples of effective amounts of polar aprotic solventsinclude, e.g., about 1% to about 95% (v/v). In some embodiments, theconcentration of polar aprotic solvent is 5% to 60% (v/v). In otherembodiments, the concentration of polar aprotic solvent is 10% to 60%(v/v). In still other embodiments, the concentration of polar aproticsolvent is 30% to 50% (v/v). Concentrations of 1% to 5%, 5% to 10%, 10%,10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or 50% to 60% (v/v) arealso suitable. In some embodiments, the polar aprotic solvent will bepresent at a concentration of 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, or 5%(v/v). In other embodiments, the polar aprotic solvent will be presentat a concentration of 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%,11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%,17.5%, 18%, 18.5%, 19%, 19.5%, or 20% (v/v).

In some embodiments, the at least one nucleic acid sequence comprisesone or more nucleic acid probes. The probes may be directly orindirectly labeled with detectable compounds such as enzymes,chromophores, fluorochromes, and haptens. DNA probes may be present atconcentrations of 0.1 to 100 μg/μL. For example, in some embodiments,DNA probes may be present at concentrations of 1 to 10 ng/μL. PNA probesmay be present at concentrations of 0.5 to 5000 nM. For example, in someembodiments, PNA probes may be present at concentrations of 5 to 1000nM.

In one embodiment, a composition of the invention comprises a mixture of40% polar aprotic solvent (v/v) (e.g., ethylene carbonate, “EC”), 10%dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, and 1-10 ng/μLprobe. Another exemplary composition of the present invention comprisesa mixture of 15% EC, 20% dextran sulfate, 600 mM NaCl, 10 mM phosphatebuffer, and 0.1 μg/μl total human DNA. Yet another exemplary compositioncomprises 15% EC, 20% dextran sulfate, 600 mM NaCl, 10 mM citric acid pH6.2, and 0.1 μg/μL non-human DNA (e.g., herring sperm, salmon sperm, orcalf thymus) OR 0.5% formamide OR 1% glycol (e.g., ethylene glycol, 1,3propanediol, or glycerol). Another exemplary composition comprises 15%EC, 20% dextran sulfate, 600 mM NaCl, 10 mM citrate buffer, pH 6.0.

(2) Polar Aprotic Solvent(s)

Different polar aprotic solvents may impart different properties on thecompositions of the invention. For example, the choice of polar aproticsolvent may contribute to the stability of the composition, sincecertain polar aprotic solvents may degrade over time. For example, thepolar aprotic solvent ethylene carbonate breaks down into ethyleneglycol, which is a relatively stable molecule, and carbon dioxide, whichcan interact with water to form carbonic acid, altering the acidity ofthe compositions of the invention. Without being bound by theory, it isbelieved that the change in pH upon breakdown of ethylene carbonate andDNA damage from long storage makes the compositions of the inventionless effective for hybridization. However, stability can be improved byreducing the pH of the composition, by adding citric acid as a buffer atpH 6.2 instead of the traditional phosphate buffer, which is typicallyused at about pH 7.4, and/or by adding ethylene glycol atconcentrations, e.g., between 0.1% to 10%, or between 0.5% to 5%, suchas, for example, 1%, 2%, 3%, etc. For example, with 10 mM citratebuffer, the compositions of the invention are stable at 2-8° C. forapproximately 8 months. Stability can also be improved if thecompositions are stored at low temperatures (e.g., −20° C.).

In addition, certain polar aprotic solvents may cause the compositionsof the invention to separate into multi-phase systems under certainconditions. The conditions under which multi-phase systems are obtainedmay be different for different polar aprotic solvents. Generally,however, as the concentration of polar aprotic solvent increases, thenumber of phases increases. For example, compositions comprising lowconcentrations ethylene carbonate (i.e., less than 20%) may exist as onephase, while compositions comprising higher concentrations of ethylenecarbonate may separate into two, or even three phases. For instance,compositions comprising 15% ethylene carbonate exist as a single phaseat room temperature, while compositions comprising 40% ethylenecarbonate consist of a viscous lower phase (approximately 25% of thetotal volume) and a less viscous upper phase (approximately 75% of thetotal volume) at room temperature.

On the other hand, some polar aprotic solvents may exist in two phasesat room temperature even at low concentrations. For example, sulfolane,γ-butyrolactone, ethylene trithiocarbonate, glycol sulfite, andpropylene carbonate exist as two phases at concentrations of 10, 15, 20,or 25% (20% dextran sulfate, 600 mM NaCl, 10 mM citrate buffer) at roomtemperature.

It may also be possible to alter the number of phases by adjusting thetemperature of the compositions of the invention. Generally, astemperature increases, the number of phases decreases. For example, at2-8° C., compositions comprising 40% ethylene carbonate may separateinto a three-phase system.

It may also be possible to alter the number of phases by adjusting theconcentration of dextran sulfate and/or salt in the composition.Generally speaking, lowering the dextran sulfate concentration(traditional concentration is 10%) and/or salt concentration may reducethe number of phases. However, depending on the particular polar aproticsolvent and its concentration in the composition, single phases may beproduced even with higher concentrations of salt and dextran sulfate.For example, a composition comprising low amounts of EC (e.g., 15%, 10%,or 5%) can work well by increasing the dextran sulfate and saltconcentrations, while still keeping a one phase system. In a particularembodiment, compositions comprising a HER2 gene DNA probe, a CEN7 PNAprobe, 15% EC, 20% dextran sulfate, 600 mM NaCl, and 10 mM phosphatebuffer are frozen at −20° C. In other embodiments, the compositions areliquid at −20° C.

Some polar aprotic solvents may produce stronger signals in one phase oranother. For example, 40% glycol sulfite produces strong signals in thelower phase and no signals in the upper phase. Similarly, certain typesof probes may produce stronger signals in one phase or another. Forexample, PNA probes tend to show stronger signals in the lower phasethan the upper phase.

Accordingly, the multiphase systems of the invention may be used toconveniently examine different aspects of a sample. For example, atwo-phase system could be used to separate samples labeled with PNAprobes from samples labeled with DNA probes. Other uses includeisolation of a specific phase exhibiting, e.g., certain advantages suchthat the isolated phase can be used as a single phase system. The probeand/or sample may be added prior to, or after isolation of a particularphase.

Hybridization applications may be performed with a one-phase compositionof the invention, with individual phases of the multiphase compositionsof the invention, or with mixtures of any one or more of the phases in amultiphase composition of the invention. For example, in a one phasesystem, a volume of the sample may be extracted for use in thehybridization. In a multiphase system, one may extract a volume ofsample from the phase of interest (e.g., the upper, lower, or middlephase) to use in the hybridization. Alternatively, the phases in amultiphase system may be mixed prior to extracting a volume of the mixedsample for use in the hybridization. However, the multiphase system mayyield strong and uneven local background staining depending on thecomposition. While, the addition of low amounts of formamide will reducebackground in a one phase system, it has little effect on a multiphasesystem with high concentrations (e.g., 40%) of a polar aprotic solvent.In addition, as the concentration of formamide increases, higherconcentrations of probe and/or longer hybridization times are requiredto maintain strong signal intensity.

(3) Optimization for Particular Applications

The compositions of the invention can be varied in order to optimizeresults for a particular application. For example, the concentration ofpolar aprotic solvent, salt, accelerating agent, blocking agent, and/orhydrogen ions (i.e. pH) may be varied in order to improve results for aparticular application.

For example, the concentration of polar aprotic solvent may be varied inorder to improve signal intensity and background staining. Generally, asthe concentration of polar aprotic solvent increases, signal intensityincreases and background staining decreases. For example, compositionscomprising 15% EC tend to show stronger signals and less background thancompositions comprising 5% EC. However, signal intensity may be improvedfor compositions having low concentrations of polar aprotic solvent(e.g., 0% to 20%) if the concentrations of salt and/or dextran sulfateare increased. For example, strong signals may be observed with 5% to10% EC when the salt concentration is raised approximately 8 to 16 timestraditional salt concentrations (i.e., approximately 1200 mM NaCl, 20 mMphosphate buffer). Likewise, as lower concentrations of polar aproticsolvent are used, higher concentrations of dextran sulfate are generallyrequired to maintain good signal and background intensity.

Accordingly, the concentrations of salt and dextran sulfate may also bevaried in order to improve signal intensity and background staining.Generally, as the concentrations of salt and dextran sulfate increase,the signal intensity increases and background decreases. For example,salt concentrations that are approximately two to four times traditionalconcentrations (i.e., 300 mM NaCl 5 mM phosphate buffer) produce strongsignals and low background. Surprisingly, however, hybridization occursusing the compositions of the invention even in the complete absence ofsalt. Signal intensities can be improved under no-salt conditions byincreasing the concentrations of accelerating agent and/or polar aproticsolvent.

Likewise, signal intensity increases as dextran sulfate concentrationincreases from 0% to 20%. However, good signals may even be observed atdextran sulfate concentrations of 0%. Signal intensity may be improvedunder low dextran sulfate conditions by increasing the polar aproticsolvent and/or salt concentrations.

In addition, the types probes used in the compositions of the inventionmay be varied to improve results. For example, in some aspects of theinvention, combinations of DNA/DNA probes may show less background thancombinations of DNA/PNA probes in the compositions of the invention orvice versa. On the other hand, PNA probes tend to show stronger signalsthan DNA probes under low salt and/or low polar aprotic solventconcentrations. In fact, PNA probes also show signals when no polaraprotic solvent is present, whereas DNA probes show weak or no signalswithout polar aprotic solvent.

D. Applications, Methods, and Uses

(1) Analytical Samples

The methods and compositions of the invention may be used fully orpartly in all types of RNA hybridization applications in the fields ofcytology, histology, or molecular biology. According to one embodiment,the RNA sequence in the methods of the invention is present in abiological sample. Examples of such samples include, e.g., tissuesamples, cell preparations, cell fragment preparations, and isolated orenriched cell component preparations. The sample may originate fromvarious tissues such as, e.g., breast, lung, colorectal, prostate, lung,head & neck, stomach, pancreas, esophagus, liver, and bladder, or otherrelevant tissues and neoplasia thereof, any cell suspension, bloodsample, fine needle aspiration, ascites fluid, sputum, peritoneum wash,lung wash, urine, feces, cell scrape, cell smear, cytospin or cytoprepcells.

The sample may be isolated and processed using standard protocols. Cellfragment preparations may, e.g., be obtained by cell homogenizing,freeze-thaw treatment or cell lysing. The isolated sample may be treatedin many different ways depending of the purpose of obtaining the sampleand depending on the routine at the site. Often the sample is treatedwith various reagents to preserve the tissue for later sample analysis,alternatively the sample may be analyzed directly. Examples of widelyused methods for preserving samples are formalin-fixed followed byparaffin-embedding and cryo-preservation.

For metaphase spreads, cell cultures are generally treated withcolcemid, or another suitable spindle pole disrupting agent, to stop thecell cycle in metaphase. The cells are then fixed and spotted ontomicroscope slides, treated with formaldehyde, washed, and dehydrated inethanol. Probes are then added and the samples are analyzed by any ofthe techniques discussed below.

Cytology involves the examination of individual cells and/or nucleicacid spreads from a biological sample. Cytological examination of asample begins with obtaining a specimen of cells, which can typically bedone by scraping, swabbing or brushing an area, as in the case ofcervical specimens, or by collecting body fluids, such as those obtainedfrom the chest cavity, bladder, or spinal column, or by fine needleaspiration or fine needle biopsy, as in the case of internal tumors. Ina conventional manual cytological preparation, the sample is transferredto a liquid suspending material and the cells in the fluid are thentransferred directly or by centrifugation-based processing steps onto aglass microscope slide for viewing. In a typical automated cytologicalpreparation, a filter assembly is placed in the liquid suspension andthe filter assembly both disperses the cells and captures the cells onthe filter. The filter is then removed and placed in contact with amicroscope slide. The cells are then fixed on the microscope slidebefore analysis by any of the techniques discussed below.

In a traditional DNA hybridization experiment using a cytologicalsample, slides containing the specimen are immersed in a formaldehydebuffer, washed, and then dehydrated in ethanol. The probes are thenadded and the specimen is covered with a coverslip. The slide isoptionally incubated at a temperature sufficient to denature anydouble-stranded nucleic acid in the specimen (e.g., 5 minutes at 82° C.)and then incubated at a temperature sufficient to allow hybridization(e.g., overnight at 45° C.). After hybridization, the coverslips areremoved and the specimens are subjected to a high-stringency wash (e.g.,10 minutes at 65° C.) followed by a series of low-stringency washes(e.g., 2×3 minutes at room temperature). The samples are then dehydratedand mounted for analysis.

In a traditional RNA hybridization experiment using cytological samples,cells are equilibrated in 40% formamide, 1×SSC, and 10 mM sodiumphosphate for 5 min, incubated at 37° C. overnight in hybridizationreactions containing 20 ng of oligonucleotide probe (e.g mix of labeled50 bp oligos), 1×SSC, 40% formamide, 10% dextran sulfate, 0.4% BSA, 20mM ribonucleotide vanadyl complex, salmon testes DNA (10 mg/ml), E. colitRNA (10 mg/ml), and 10 mM sodium phosphate. Then washed twice with4×SSC/40% formamide and again twice with 2×SSC/40% formamide, both at37° C., and then with 2×SSC three times at room temperature.Digoxigenin-labeled probes can then e.g. be detected by using amonoclonal antibody to digoxigenin conjugated to Cy3. Biotin-labeledprobes can then e.g. be detected by using streptavidin-Cy5. Detectioncan be by fluorescence or CISH.

Histology involves the examination of cells in thin slices of tissue. Toprepare a tissue sample for histological examination, pieces of thetissue are fixed in a suitable fixative, typically an aldehyde such asformaldehyde or glutaraldehyde, and then embedded in melted paraffinwax. The wax block containing the tissue sample is then cut on amicrotome to yield thin slices of paraffin containing the tissue,typically from 2 to 10 microns thick. The specimen slice is then appliedto a microscope slide, air dried, and heated to cause the specimen toadhere to the glass slide. Residual paraffin is then dissolved with asuitable solvent, typically xylene, toluene, or others. These so-calleddeparaffinizing solvents are then removed with a washing-dehydratingtype reagent prior to analysis of the sample by any of the techniquesdiscussed below. Alternatively, slices may be prepared from frozenspecimens, fixed briefly in 10% formalin or other suitable fixative, andthen infused with dehydrating reagent prior to analysis of the sample.

In a traditional DNA hybridization experiment using a histologicalsample, formalin-fixed paraffin embedded tissue specimens are cut intosections of 2-6 μm and collected on slides. The paraffin is melted(e.g., 30-60 minutes at 60° C.) and then removed (deparaffinated) bywashing with xylene (or a xylene substitute), e.g., 2×5 minutes. Thesamples are rehydrated, washed, and then pre-treated (e.g., 10 minutesat 95-100° C.). The slides are washed and then treated with pepsin oranother suitable permeabilizer, e.g., 3-15 minutes at 37° C. The slidesare washed (e.g., 2×3 minutes), dehydrated, and probe is applied. Thespecimens are covered with a coverslip and the slide is optionallyincubated at a temperature sufficient to denature any double-strandednucleic acid in the specimen (e.g. 5 minutes at 82° C.), followed byincubation at a temperature sufficient to allow hybridization (e.g.,overnight at 45° C.). After hybridization, the coverslips are removedand the specimens are subjected to a high-stringency wash (e.g., 10minutes at 65° C.) followed by a series of low-stringency washes (e.g.,2×3 minutes at room temperature). The samples are then dehydrated andmounted for analysis.

In a traditional RNA hybridization experiment using a histologicalsample, slides with FFPE tissue sections are deparaffinized in xylenefor 2×5 min, immerged in 99% ethanol 2×3 min, in 96% ethanol 2×3 min,and then in pure water for 3 min. Slides are placed in a humiditychamber, Proteinase K is added, and slides are incubated at RT for 5min-15 min. Slides are immersed in pure water for 2×3 min, immersed in96% ethanol for 10 sec, and air-dried for 5 min. Probes are added to thetissue section and covered with coverslip. The slides are incubated at55° C. in humidity chamber for 90 min. After incubation, the slides areimmersed in a Stringent Wash solution at 55° C. for 25 min, and thenimmersed in TBS for 10 sec. The slides are incubated in a humiditychamber with antibody for 30 min. The slides are immersed in TBS for 2×3min, then in pure water for 2×1 min, and then placed in a humiditychamber. The slides are then incubated with substrate for 60 min, andimmersed in tap water for 5 min.

In a traditional northern blot procedure, the RNA target sample isdenatured for 10 minutes at 65° C. in RNA loading buffer and immediatelyplaced on ice. The gels are loaded and electrophoresed with 1×MOPSbuffer (10×MOPS contains 200 mM morpholinopropansulfonic acid, 50 mMsodium acetate, 10 mM EDTA, pH 7.0) at 25 V overnight. The gel is thenpre-equilibrated in 20×SSC for 10 min and the RNA is transferred to anylon membrane using sterile 20×SSC as transfer buffer. The nucleicacids are then fixed on the membrane using, for example, UV-crosslinking at 120 mJ or baking for 30 min at 120° C. The membrane is thenwashed in water and air dried. The membrane is placed in a sealableplastic bag and prehybridized without probe for 30 min at 68° C. Theprobe is denatured for 5 min at 100° C. and immediately placed on ice.Hybridization buffer (prewarmed to 68° C.) is added and the probe ishybridized at 68° C. overnight. The membrane is then removed from thebag and washed twice for 5 min each with shaking in a low stringencywash buffer (e.g., 2×SSC, 0.1% SDS) at room temperature. The membrane isthen washed twice for 15 min each in prewarmed high stringency washbuffer (e.g., 0.1×SSC, 0.1% SDS) at 68° C. The membrane may then bestored or immediately developed for detection.

Additional examples of traditional hybridization techniques can befound, for example, in Sambrook et al., Molecular Cloning A LaboratoryManual, 2^(nd) Ed., Cold Spring Harbor Laboratory Press, (1989) atsections 1.90-1.104, 2.108-2.117, 4.40-4.41, 7.37-7.57, 8.46-10.38,11.7-11.8, 11.12-11.19, 11.38, and 11.45-11.57; and in Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1998)at sections 2.9.1-2.9.6, 2.10.4-2.10.5, 2.10.11-2.10.16, 4.6.5-4.6.9,4.7.2-4.7.3, 4.9.7-4.9.15, 5.9.18, 6.2-6.5, 6.3, 6.4, 6.3.3-6.4.9,5.9.12-5.9.13, 7.0.9, 8.1.3, 14.3.1-14.3.4, 14.9, 15.0.3-15.0.4,15.1.1-15.1.8, and 20.1.24-20.1.25.

(2) Hybridization Techniques

The compositions and methods of the present invention can be used fullyor partly in all types of RNA hybridization techniques known in the artfor cytological and histological samples. Such techniques include, forexample, in situ hybridization (ISH), fluorescent in situ hybridization(FISH; including multi-color FISH, Fiber-FISH, etc.), chromogenic insitu hybridization (CISH), silver in situ hybridization (SISH), andarrays. The compositions of the invention will improve the efficiency oftraditional RNA hybridization applications, e.g., by reducing thedenaturation and hybridization temperatures and/or the time.

Molecular probes that are suitable for use in the hybridizations of theinvention are described, e.g., in U.S. Patent Publication No.2005/0266459, which is incorporated herein by reference. In general,probes may be prepared by chemical synthesis, PCR, or by amplifying aspecific DNA sequence by cloning, inserting the DNA into a vector, andamplifying the vector an insert in appropriate host cells. Commonly usedvectors include bacterial plasmids, cosmids, bacterial artificialchromosomes (BACs), PI diverted artificial chromosomes (PACs), or yeastartificial chromosomes (YACs). The amplified DNA is then extracted andpurified for use as a probe. Methods for preparing and/or synthesizingprobes are known in the art, e.g., as disclosed in PCT/US02/30573.

The nucleic acid probe may be a double or single stranded nucleic acidfragment or sequence, such as a DNA, RNA, or analogs such as PNA or LNA.The probes may be labeled to make identification of the probe-targethybrid possible by use, for example, of a fluorescence or bright fieldmicroscope/scanner. In some embodiments, the probe may be labeled usingradioactive labels such as ³¹P, ³³P, or ³²S, non-radioactive labels suchas digoxigenin and biotin, or fluorescent labels

In general, the type of probe determines the type of feature one maydetect in a hybridization assay. For example, large insert probes may beused to target unique single-copy sequences. With these large probes,the hybridization efficiency is inversely proportional to the probesize. Smaller probes can be used to detect aberrations such asdeletions, amplifications, inversions, duplications, and aneuploidy. Forexample, differently-colored locus-specific probes can be used to detecttranslocations via split-signal in situ hybridization.

In general, the ability to discriminate between closely relatedsequences is inversely proportional to the length of the hybridizationprobe because the difference in thermal stability decreases between wildtype and mutant complexes as probe length increases. Probes of greaterthan 10 bp in length are generally required to obtain the sequencediversity necessary to correctly identify a unique organism or clinicalcondition of interest. On the other hand, sequence differences as subtleas a single base (point mutation) in very short oligomers (<10 basepairs) can be sufficient to enable the discrimination of thehybridization to complementary nucleic acid target sequences as comparedwith non-target sequences.

In one embodiment, at least one set of the hybridization probes maycomprise one or more PNA probes, as defined above and as described inU.S. Pat. No. 7,105,294, which is incorporated herein by reference.Methods for synthesizing PNA probes are described in PCT/US02/30573.Alternatively, or in addition, at least one set of the hybridizationprobes in any of the techniques discussed above may comprise one or morelocked nucleic acid (LNA) probes, as described in WO 99/14226, which isincorporated herein by reference. Due to the additional bridging bondbetween the 2′ and 4′ carbons, the LNA backbone is pre-organized forhybridization. LNA/RNA interactions are stronger than the correspondingDNA/RNA interactions, as indicated by a higher melting temperature.Thus, the compositions and methods of the invention, which decrease theenergy required for hybridization, are particularly useful forhybridizations with LNA probes.

In one embodiment, the probes may comprise a detectable label (amolecule that provides an analytically identifiable signal that allowsthe detection of the probe-target hybrid), as described in U.S. PatentPublication No. 2005/0266459, which is incorporated herein by reference.The detectable label may be directly attached to a probe, or indirectlyattached to a probe, e.g., by using a linker. Any labeling method knownto those in the art, including enzymatic and chemical processes, can beused for labeling probes used in the methods and compositions of theinvention. In other embodiments, the probes are not labeled.

In general, in situ hybridization techniques such as FISH, CISH, andSISH for DNA detection, employ large, mainly unspecified, nucleic acidprobes that hybridize with varying stringency. Using large probesrenders the in situ hybridization technique very sensitive. However, thesuccessful use of large probes in traditional hybridization assaysdepends on blocking the undesired background staining derived from,e.g., repetitive sequences that are present throughout the genome.Traditional methods for decreasing nonspecific probe binding includesaturating the binding sites on proteins and tissue by incubating tissuewith prehybridization solutions containing ficoll, bovine serum albumin(BSA), polyvinyl pyrrolidone, and nucleic acids. Such blocking steps aretime-consuming and expensive. As discussed below, the methods andcompositions of the invention advantageously reduce and/or eliminate theneed for such blocking steps and blocking reagents. However, in oneembodiment, repetitive sequences may be suppressed according to themethods known in the art, e.g., as disclosed in PCT/US02/30573.

Bound probes may be detected in cytological and histological sampleseither directly or indirectly with fluorochromes (e.g., FISH), organicchromogens (e.g., CISH), silver particles (e.g., SISH), or othermetallic particles (e.g., gold-facilitated fluorescence in situhybridization, GOLDFISH). Thus, depending on the method of detection,populations of cells obtained from a sample to be tested may bevisualized via fluorescence microscopy or conventional brightfield lightmicroscopy.

Hybridization assays on cytological and histological samples areimportant tools for determining the number, size, and/or location ofspecific RNA sequences.

FISH is typically used when multiple color imaging is required and/orwhen the protocol calls for quantification of signals. The techniquegenerally entails preparing a cytological sample, labeling probes,optionally denaturing the target and the probe, hybridizing the probe tothe target sequence, and detecting the signal. Typically, thehybridization reaction fluorescently stains the targeted sequences sothat their location, size, or number can be determined usingfluorescence microscopy, flow cytometry, or other suitableinstrumentation. RNA sequences ranging from megabases down to severalkilobases can be studied using FISH. With enhanced fluorescencemicroscope techniques, such as, for example, deconvolution, even asingle mRNA molecule can be detected. FISH may also be used on metaphasespreads and interphase nuclei.

One of the most important applications for FISH has been in detectingsingle-copy sequences, in particular disease related sequences in humansand other eukaryotic model species, and the detection of infectionsagents. FISH may be used to detect, e.g., chromosomal aneuploidy inprenatal diagnoses, hematological cancers, and solid tumors; geneabnormalities such as oncogene amplifications, gene deletions, or genefusions; translocations, duplications, insertions, or inversions;contiguous gene syndromes such as microdeletion syndrome; the geneticeffects of various therapies; viral nucleic acids in somatic cells andviral integration sites in chromosomes; etc. FISH techniques includemultiplex FISH (m-FISH), spectral karyotyping (SKY), combined binaryration labeling (COBRA), color-changing karyotyping, cross-species colorbanding, high resolution multicolor banding, telomeric multiplex FISH(TM-FISH), split-signal FISH (ssFISH), and fusion-signal FISH.

CISH and SISH may be used for many of the same applications as FISH, andhave the additional advantage of allowing for analysis of the underlyingtissue morphology, for example in histopathology applications. If FISHis performed, the hybridization mixture may contain sets of distinct andbalanced pairs of probes, as described in U.S. Pat. No. 6,730,474, whichis incorporated herein by reference. For CISH, the hybridization mixturemay contain at least one set of probes configured for detection with oneor more conventional organic chromogens, and for SISH, the hybridizationmixture may contain at least one set of probes configured for detectionwith silver particles, as described in Powell R D et al.,“Metallographic in situ hybridization,” Hum. Pathol., 38:1145-59 (2007).

The compositions of the invention may also be used fully or partly inall types of molecular biology techniques involving hybridization,including blotting and probing (e.g., northern blotting, etc.), andarrays.

(3) Hybridization Conditions

The method of the present invention involves the use of polar aproticsolvents in RNA hybridization applications. The compositions of thepresent invention are particularly useful in said method.

Hybridization methods using the compositions of the invention mayinvolve applying the compositions to a sample comprising a target RNAsequence. The polar aprotic solvent will interact with the probe and thetarget and facilitate the annealing of the probe to the target. Thepolar aprotic solvent will also facilitate the denaturation of anydouble stranded sequences, if necessary, to secure access for the probeto hybridize with the target sequence. The polar aprotic solventsspecified in the present invention speed up the hybridization processconsiderably and reduce the harshness and toxicity of the hybridizationconditions compared to traditional formamide-containing buffers.

Hybridizations using the compositions of the invention may be performedusing the same assay methodology as for RNA hybridizations performedwith traditional compositions. For example, the heat pre-treatment,digestion, denaturation, hybridization, washing, and mounting steps mayuse the same conditions in terms of volumes, temperatures, reagents andincubation times as for traditional compositions. However, thecompositions of the invention allow for shorter hybridization times.

A great variation exists in the traditional RNA hybridization protocolsknown in the art. The compositions of the invention may be used in anyof the traditional hybridization protocols known in the art.

Alternatively, assays using the compositions of the invention can bechanged and optimized from traditional methodologies, for example, bydecreasing the hybridization time, increasing or decreasing thehybridization temperatures, and/or increasing or decreasing thehybridization volumes.

For example, in some embodiments, the compositions of the invention willproduce strong signals when the hybridization temperature is from 20 to60° C. In other embodiments, the compositions of the invention willproduce strong signals when the hybridization temperature is from 20 to30° C., 30 to 40° C., 40 to 50° C., or 50 to 60° C. In otherembodiments, the compositions of the invention will produce strongsignals when the hybridization temperature is 37, 40, 45, or 50° C.

In other embodiments, the compositions of the invention will producestrong signals when the hybridization time is from 0 minutes to 24hours. In other embodiments, the compositions of the invention willproduce strong signals when the hybridization time is from 0 minute to 8hours. In other embodiments, the compositions of the invention willproduce strong signals when the hybridization time is 0 minutes, 5minutes, 15 minutes, 30 minutes, 60 minutes, 180 minutes, or 240minutes.

Accordingly, hybridizations using the compositions of the invention maybe performed in less than 8 hours. In other embodiments, thehybridization step is performed in less than 6 hours. In still otherembodiments, the hybridization step is performed within 4 hours. Inother embodiments, the hybridization step is performed within 3 hours.In yet other embodiments, the hybridization step is performed within 2hours. In other embodiments, the hybridization step is performed within1 hour. In still other embodiments, the hybridization step is performedwithin 30 minutes. In other embodiments, the hybridization step can takeplace within 15 minutes. The hybridization step can even take placewithin 10 minutes or in less than 5 minutes. FIGS. 1 and 2 illustrate atypical time-course for DNA hybridization applications performed onhistological and cytological samples, respectively, using thecompositions of the invention compared to hybridization applicationsusing a traditional compositions.

If the hybridization applications of the invention include an optionaldenaturation step, the denaturation temperature may be from 60 to 100°C. In other embodiments, the compositions of the invention will producestrong signals when the denaturation temperature is from 60 to 70° C.,70 to 80° C., 80 to 90° C. or 90 to 100° C. In other embodiments, thecompositions of the invention will produce strong signals when thedenaturation temperature is 72, 82, or 92° C. The denaturation time maybe from 0 to 10 minutes. In other embodiments, the compositions of theinvention will produce strong signals when the denaturation time is from0 to 5 minutes. In other embodiments, the compositions of the inventionwill produce strong signals when the denaturation time is 0, 1, 2, 3, 4,or 5 minutes. It will be understood by those skilled in the art that inmost cases, RNA detection does not require a denaturation step.

The concentration of probe may also be varied in order to produce strongsignals and/or reduce background. For example, as hybridization timedecreases, the amount of probe may be increased in order to improvesignal intensity. On the other hand, as hybridization time decreases,the amount of probe may be decreased in order to improve backgroundstaining.

The compositions of the invention also eliminate the need for a blockingstep during hybridization applications by improving signal andbackground intensity by blocking the binding of, e.g., repetitivesequences to the target DNA. Thus, there is no need to use total humanDNA, blocking-PNA, COT-1 DNA, or DNA from any other source as a blockingagent. However, background levels can be further reduced by addingagents that reduce non-specific binding, such as to the cell membrane,such as small amounts of total human DNA or non-human-origin DNA (e.g.,salmon sperm DNA) to a hybridization reaction using the compositions ofthe invention.

The aqueous compositions of the invention furthermore provide for thepossibility to considerably reduce the concentration of nucleic acidsequences included in the composition. Generally, the concentration ofprobes may be reduced from 2 to 8-fold compared to traditionalconcentrations. For example, if HER2 DNA probes and CEN17 PNA probes areused in the compositions of the invention, their concentrations may bereduced by ¼ and ½, respectively, compared to their concentrations intraditional hybridization compositions. This feature, along with theabsence of any requirement for blocking DNA, such as blocking-PNA orCOT1, allows for an increased probe volume in automated instrumentsystems compared to the traditional 10 μL volume used in traditionalcompositions systems, which reduces loss due to evaporation, asdiscussed in more detail below.

Reducing probe concentration also reduces background. However, reducingthe probe concentration is inversely related to the hybridization time,i.e., the lower the concentration, the higher hybridization timerequired. Nevertheless, even when extremely low concentrations of probeare used with the aqueous compositions of the invention, thehybridization time is still shorter than with traditional compositions.

The compositions of the invention often allow for better signal-to-noiseratios than traditional hybridization compositions. For example, withcertain probes, a one hour hybridization with the compositions of theinvention will produce similar background and stronger signals than anovernight hybridization in a traditional compositions. Background is notseen when no probe is added.

Traditional assay methods may also be changed and optimized when usingthe compositions of the invention depending on whether the system ismanual, semi-automated, or automated. For example, a semi-automated or afully automated system will benefit from the short hybridization timesobtained with the compositions of the invention. The short hybridizationtime may reduce the difficulties encountered when traditionalcompositions are used in such systems. For example, one problem withsemi-automated and fully automated systems is that significantevaporation of the sample can occur during hybridization, since suchsystems require small sample volumes (e.g., 10-150 μL), elevatedtemperatures, and extended hybridization times (e.g., 14 hours). Thus,proportions of the components in traditional hybridization compositionsare fairly invariable. However, since the compositions of the inventionallow for faster hybridizations, evaporation is reduced, allowing forincreased flexibility in the proportions of the components inhybridization compositions used in semi- and automated systems.

For example, two automated instruments have been used to performhybridizations to DNA targets using the compositions of the invention.Compositions comprising 40% ethylene carbonate (v/v) have been used inthe apparatus disclosed in PCT application DK2008/000430, andcompositions comprising 15% ethylene carbonate (v/v) have been used inthe HYBRIMASTER HS-300 (Aloka CO. LTD, Japan). When the compositions ofthe invention are used in the HYBRIMASTER HS-300, the instrument canperform rapid FISH hybridization with water in place of the traditionaltoxic formamide mix, thus improving safety and reducing evaporation. Ifwater wetted strips are attached to the lid of the inner part of theAloka instrument's reaction unit (hybridization chamber), e.g., asdescribed in U.S. patent application Ser. No. 11/031,514, which isincorporated herein by reference, evaporation is reduced even further.

Another problem with automated imaging analysis is the number of imagesneeded, the huge amount of storage place required, and the time requiredto take the images. The compositions of the invention address thisproblem by producing very strong signals compared to traditionalcompositions. Because of the very strong signals produced by thecompositions of the invention, the imaging can be done at lowermagnification than required for traditional compositions and can stillbe detected and analyzed, e.g., by algorithms. Since the focal planebecomes wider with lower magnification, the compositions of theinvention reduce or eliminate the requirement to take serial sections ofa sample. As a result, the overall imaging is much faster, since thecompositions of the invention require fewer or no serial sections andeach image covers much greater area. In addition, the overall time foranalysis is faster, since the total image files are much smaller.

Thus, the compositions and methods of the invention solve many of theproblems associated with traditional hybridization compositions andmethods.

The disclosure may be understood more clearly with the aid of thenon-limiting examples that follow, which constitute preferredembodiments of the compositions according to the disclosure. Other thanin the examples, or where otherwise indicated, all numbers expressingquantities of ingredients, reaction conditions, and so forth used in thespecification and claims are to be understood as being modified in allinstances by the term “about.” Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the followingspecification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained herein. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should be construed in light of the number of significantdigits and ordinary rounding approaches.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope are approximations, the numerical values set forth inthe specific example are reported as precisely as possible. Anynumerical value, however, inherently contains certain errors necessarilyresulting from the standard deviation found in its respective testingmeasurements. The examples that follow illustrate the present inventionand should not in any way be considered as limiting the invention.

EXAMPLES

Reference will now be made in detail to specific embodiments of theinvention. While the invention will be described in conjunction withthese embodiments, it will be understood that they are not intended tolimit the invention to those embodiments. On the contrary, the inventionis intended to cover alternatives, modifications, and equivalents, whichmay be included within the invention as defined by the appended claims.

The reagents used in the following examples are from Dako's HistologyFISH Accessory Kit (K5599) and Cytology FISH Accessory Kit (K5499) (DakoDenmark A/S, Glostrup Denmark). The kits contain all the key reagents,except for probe, required to complete a FISH procedure forformalin-fixed, paraffin-embedded tissue section specimens. All sampleswere prepared according to the manufacturer's description. The DakoHybridizer (S2451, Dako) was used for the digestion, denaturation, andhybridization steps.

Evaluation of FISH slides was performed within a week afterhybridization using a Leica DM6000B fluorescence microscope, equippedwith DAN, FITC, Texas Red single filters and FITC/Texas Red doublefilter under 10×, 20×, 40×, and 100× oil objective.

Evaluation of CISH slides was performed using an Olympus BX51 lightmicroscope, under 4×, 10×, 20×, 40×, and 60× objective.

In the Examples that follow, “dextran sulfate” refers to the sodium saltof dextran sulfate (D8906, Sigma) having a molecular weightM_(w)>500,000. All concentrations of polar aprotic solvents are providedas v/v percentages. Phosphate buffer refers to a phosphate bufferedsolution containing NaH₂PO₄, 2H₂O (sodium phosphate dibasic dihydrate)and Na₂HPO₄, H₂O (sodium phosphate monobasic monohydrate). Citratebuffer refers to a citrate buffered solution containing sodium citrate(Na₃C₆H₅O₇, 2H₂O; 1.06448, Merck) and citric acid monohydrate (C₆H₈O₇,H₂O; 1.00244, Merck).

General histology FISH/CISH Procedure for Examples 1-20

The slides with cut formalin-fixed paraffin embedded (FFPE) multipletissue array sections from humans (tonsils, mammacarcinoma, kidney andcolon) were baked at 60° C. for 30-60 min, deparaffinated in xylenebaths, rehydrated in ethanol baths and then transferred to Wash Buffer.The samples were then pre-treated in Pre-Treatment Solution at a minimumof 95° C. for 10 min and washed 2×3 min. The samples were then digestedwith Pepsin RTU at 37° C. for 3 min, washed 2×3 min, dehydrated in aseries of ethanol evaporations, and air-dried. The samples were thenincubated with 10 μL FISH probe as described under the individualexperiments. The samples were then washed by Stringency Wash at 65° C.10 min, then washed 2×3 min, then dehydrated in a series of ethanolevaporations, and air-dried. Finally, the slides were mounted with 15 μLAntifade Mounting Medium. When the staining was completed, observerstrained to assess signal intensity, morphology, and background of thestained slides performed the scoring.

General Cytology FISH Procedure for Examples 21-22

Slides with metaphases preparation were fixed in 3.7% formaldehyde for 2min, washed 2×5 min, dehydrated in a series of ethanol evaporations, andair-dried. The samples were then incubated with 10 μL FISH probe asdescribed under the individual experiments. The samples were then washedby Stringency Wash at 65° C. 10 min, then washed 2×3 min, thendehydrated in a series of ethanol evaporations, and air-dried. Finally,the slides were mounted with 15 μL Antifade Mounting Medium. When thestaining was completed, observers trained to assess signal intensity andbackground of the stained slides performed the scoring as described inthe scoring for guidelines for tissue sections.

Histology CISH Procedure for Example 23

The reagents used in Example 23 are from Dako's PNA ISH Detection Kitfor fluorescein-labeled PNA probes (K5201) (Dako Denmark A/S, GlostrupDenmark). The kit contains all reagents necessary for performing an insitu hybridization, except for the specific probe. All samples wereprepared according to the manufacturer's description. The DakoHybridizer (S2451, Dako) was used as indicated.

Slides with tissue sections were immersed in xylene for 2×5 min, then in99% ethanol for 2×3 min, then in 96% ethanol for 2×3 min, and then inpure water for 3 min. The slides were then placed in a humidity chamberand 200 μL Proteinase K was added. The samples that were designated toreceive PNA probes were incubated at RT for 5 min, and the samples thatwere designated to receive LNA probes were incubated at RT for 15 min.The slides were then immersed in pure water for 2×3 min, immersed in 96%ethanol for 10 sec, and air-dried for 5 min. Fluorescein-conjugated PNAor LNA probe (85 μL) was added to the tissue section and the slides werecovered with coverslip. The samples with PNA probe were incubated at 55°C., and the samples with LNA probe were incubated at 42° C. (Hybridizer,S2451). After incubation, the coverslips were removed, the slides wereimmersed in a Stringent Wash solution at 55° C. (for PNA probes) or 30°C. (for LNA probes) for 25 min, and then immersed in TBS for 10 sec. Theslides were transferred to a humidity chamber and five to six drops ofthe Anti-FITC/AP antibody was added. The slides were incubated for 30min., the antibody was tapped off, the slides were immersed in TBS for2×3 min, then in pure water for 2×1 min, and then placed in a humiditychamber. Five to six drops of the BCIP/NBT/levamisole chromogenicsubstrate was added to the sample, the slides were incubated for 60 min,the substrate was tapped off, and the slides were immersed in tap waterfor 5 min. The slides were then counterstained with Dako Nuclear FastRed (S1963) for 1 min and mounted in Tissue-Mount (Sakura, TheNetherlands).

Histology CISH Procedure for Example 24 and 25

The reagents used in Example 24 are from Dako's PNA ISH Detection Kitfor fluorescein-labeled PNA probes (K5201) (Dako Denmark A/S, GlostrupDenmark). The kit contains all reagents necessary for performing an insitu hybridization, except for the specific probe. All samples wereprepared according to the manufacturer's description. The DakoHybridizer (S2451, Dako) was used as indicated.

Slides with tissue sections were immersed in xylene for 2×5 min, then in99% ethanol for 2×3 min, then in 96% ethanol for 2×3 min, and then inRNase-free water for 3 min. The slides were then placed in a humiditychamber and 200 μL Proteinase K was added, and the slides were incubatedat RT for 5 min. The slides were then immersed in RNase-free water for2×3 min, immersed in 96% ethanol for 10 sec, and air-dried for 5 min.Fluorescein-conjugated PNA or LNA probe (50 μL) was added to the tissuesection and the slides were covered with a coverslip and sealed withCoverslip Sealant (K5999, Dako). The samples were incubated at 35° C. or45° C. in a Hybridizer (S2451, Dako) for 90 min. After incubation, thecoverslips were removed, the slides were immersed in a Stringent Washsolution at 35° C. or 45° C. for 25 min, and then immersed in TBS. Theslides were transferred to a Dako Autostainer (S3400, Dako), and thestaining was performed using two drop zones of 2×200 μL with thefollowing staining protocol: Rinse TBS, Anti-FITC/AP antibody incubationfor 30 min, 2× rinse in TBS, 2× rinse in pure water, BCIP/NBT substratefor 30 min, rinse in TBS, BCIP/NBT substrate for 30 min, rinse in purewater, Nuclear Fast Red (S1963) 1 min, and 6× rinse in pure water. Theslides were then removed from the Dako Autostainer and mounted inTissue-Mount (Sakura, The Netherlands).

Scoring Guidelines of Tissue Sections

The signal/staining intensities were evaluated on a 0-3 scale with 0meaning no signal and 3 equating to a strong signal. The cell/tissuestructures are evaluated on a 0-3 scale with 0 meaning no structure andno nuclei boundaries and 3 equating to intact structure and clear nucleiboundaries. Between 0 and 3 there are additional grades 0.5 apart fromwhich the observer can assess signal intensity, tissue structure, andbackground.

The signal/staining intensity is scored after a graded system on a 0-3scale.

-   -   0 No signal/staining is seen.    -   1 The signal/staining intensity is weak.    -   2 The signal/staining intensity is moderate.    -   3 The signal/staining intensity is strong.

The scoring system allows the use of ½ grades.

The tissue and nuclear structure is scored after a graded system on a0-3 scale.

-   -   0 The tissue structures and nuclear borders are completely        destroyed.    -   1 The tissue structures and/or nuclear borders are poor. This        grade includes situations where some areas have empty nuclei.    -   2 Tissue structures and/or nuclear borders are seen, but the        nuclear borders are unclear. This grade includes situations        where a few nuclei are empty.    -   3 Tissue structures and nuclear borders are intact and clear.

The scoring system allows the use of ½ grades.

The background is scored after a graded system on a 0-3 scale.

-   -   0 Little to no background is seen.    -   1 Some background.    -   2 Moderate background.    -   3 High Background.

The scoring system allows the use of ½ grades.

Example 1

This example compares the signal intensity and cell morphology fromsamples treated with the compositions of the invention or traditionalhybridization solutions as a function of denaturation temperature.

FISH Probe composition I: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% formamide (15515-026, Invitrogen), 5 μM blockingPNAs (see Kirsten Yang Nielsen et al., PNA Suppression Method Combinedwith Fluorescence In Situ Hybridisation (FISH) Technique inPRINS and PNATechnologies in Chromosomal Investigation, Chapter 10 (Franck Pellestored.) (Nova Science Publishers, Inc. 2006)), 10 ng/μL Texas Red labeledCCND1 gene DNA probe (RP11-1143E20, size 192 kb).

FISH Probe composition II: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate (03519, Fluka), 5 μM blockingPNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe (RP11-1143E20,size 192 kb).

Phases of different viscosity, if present, were mixed before use. TheFISH probes were denatured as indicated for 5 min and hybridized at 45°C. for 60 minutes.

Results:

Signal (I) (II) Cell morphology Denaturation temperature Formamide ECFormamide EC 72° C. 0 2 Good Good 82° C. ½ 3 Good Good 92° C. ½ 3 Notgood Not good Signals scored as “3” were clearly visible in a 20xobjective.

Example 2

This example compares the signal intensity and background staining fromsamples treated with the compositions of the invention or traditionalhybridization solutions as a function of hybridization time.

FISH Probe composition I: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% formamide, 5 μM blocking PNAs, 10 ng/μL Texas Redlabeled CCND1 gene DNA probe.

FISH Probe composition II: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate, 5 μM blocking PNAs, 10 ng/μLTexas Red labeled CCND1 gene DNA probe.

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 14hours, 4 hours, 2 hours, 60 minutes, 30 minutes, 15 minutes, 0 minutes.

Results:

Signal (I) (II) Background staining Hybridization time Formamide ECFormamide EC 14 hours 3 3 +½ +2  4 hours 1 3 +½ +1  2 hours ½ 3 +0 +1 60min. ½ 3 +0 +1 30 min. 0 2½ +0 +1 15 min. 0 2 +0 +1  0 min. 0 1 +0 +½Signals scored as “3” were clearly visible in a 20x objective.

Example 3

This example compares the signal intensity from samples treated with thecompositions of the invention having different polar aprotic solvents ortraditional hybridization solutions.

FISH Probe composition I: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% formamide, 5 μM blocking PNAs, 10 ng/μL Texas Redlabeled CCND1 gene DNA probe.

FISH Probe composition II: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate (EC), 5 μM blocking PNAs, 10ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe composition III: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Propylene carbonate (PC) (540013, Aldrich), 5 μMblocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe composition IV: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Sulfolane (SL) (T22209, Aldrich), 5 μM blockingPNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe composition V: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Aceto nitrile (AN) (C02CIIX, Lab-Scan), 5 μMblocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe composition VI: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% γ-butyrolactone (GBL) (B103608, Aldrich), 5 μMblocking PNAs, 7.5 ng/μL Texas Red labeled CCND1 gene DNA probe.

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes.

Results:

Signal (I) (II) (III) (IV) (V) (VI) Formamide EC PC SL AN GBL ½ 3 3 3 23 Signals scored as “3” were clearly visible in a 20x objective.

Example 4

This example compares the signal intensity from samples treated with thecompositions of the invention having different concentrations of polaraprotic solvent.

FISH Probe Compositions: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 10-60% Ethylene carbonate (as indicated), 5 μMblocking PNAs, 7.5 ng/μL Texas Red labeled IGK-constant DNA gene probe((CTD-3050E15, RP11-1083E8; size 227 kb) and 7.5 ng/μL FITC labeledIGK-variable gene DNA probe (CTD-2575M21, RP11-122B6, RP11-316G9; size350 and 429 kb).

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes.

Results:

Ethylene carbonate (EC) 10% 20% 30% 40% 60% Signal Texas Red 1½ 2 3 3 2intensity FITC 1 1½ 2 2½ 2 Signals scored as “3” were clearly visible ina 20x objective.

Example 5

This example compares the signal intensity and background intensity fromsamples treated with the compositions with and without PNA blocking.

FISH Probe Compositions: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate, 7.5 ng/μL Texas Red labeledCCND1 gene DNA probe.

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes.

Results:

Ethylene carbonate (EC) PNA- blocking Non- PNA blocking Signal intensity3 3 Background intensity ½+ ½+ Signals scored as “3” were clearlyvisible in a 20x objective.

Example 6

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of probe concentration andhybridization time.

FISH Probe Compositions: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% Ethylene carbonate, and 10, 7.5, 5 or 2.5 ng/μLTexas Red labeled CCND1 gene DNA probe (as indicated).

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 3hours, 2 hours and 1 hours.

Results:

Signal Intensity Hybridization (I) (II) (III) (IV) time 10 ng/μL 7.5ng/μL 5 ng/μL 2.5 ng/μL 3 hours 3 3 3 3 2 hours 3 3 3 1 1 hours 3 3 3 ½Signals scored as “3” were clearly visible in a 20x objective.

Example 7

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of salt, phosphate, andbuffer concentrations.

FISH Probe Compositions: 10% dextran sulfate, ([NaCl], [phosphatebuffer], [TRIS buffer] as indicated in Results), 40% Ethylene carbonate,7.5 ng/μL Texas Red labeled CCND1 gene DNA probe.

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes.

Results:

[NaCl] 300 mM 100 mM 0 mM Signal intensity 2 1 ½ phosphate [0 mM] Signalintensity 3 2½ ½ phosphate [5 mM] Signal intensity — — 3 phosphate [35mM] Signal intensity — — 2 TRIS [40 mM] Signals scored as “3” wereclearly visible in a 20x objective.

Example 8

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of dextran sulfateconcentration.

FISH Probe Compositions: 0, 1, 2, 5, or 10% dextran sulfate (asindicated), 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate,5 ng/μL Texas Red labeled SIL-TAL1 gene DNA probe (RP1-278O13; size 67kb) and 6 ng/μL FITC SIL-TAL1 (ICRFc112-112C1794, RP11-184J23, RP11-8J9,CTD-2007B18, 133B9; size 560 kb).

Phases of different viscosity, if present, were mixed before use. TheFISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60minutes. No blocking.

Results:

Signal Intensity % Dextran Sulfate Texas Red Probe FITC Probe  0% 1 1 1% 1 1  2% 1½ 1½  5% 2 2½ 10% 2 2½ NOTE: this experiment did notproduce results scored as “3” because the SIL-TAL1 Texas Red labeledprobe is only 67 kb and was from a non-optimized preparation.

Example 9

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of dextran sulfate, salt,phosphate, and polar aprotic solvent concentrations.

FISH Probe Composition Ia: 34% dextran sulfate, 0 mM NaCl, 0 mMphosphate buffer, 0% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition Ib: 34% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 0% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition Ic: 34% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, 0% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition IIa: 32% dextran sulfate, 0 mM NaCl, 0 mMphosphate buffer, 5% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition IIb: 32% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 5% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition IIc: 32% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, 5% ethylene carbonate, 10 ng/μL Texas Red labeled HER2gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.

FISH Probe Composition IIIc: 30% dextran sulfate, 0 mM NaCl, 0 mMphosphate buffer, 10% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition IIIb: 30% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 10% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition IIIc: 30% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, 10% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition IVa: 28% dextran sulfate, 0 mM NaCl, 0 mMphosphate buffer, 15% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition IVb: 28% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 15% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Composition IVc: 28% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, 15% ethylene carbonate, 10 ng/μL Texas Red labeledHER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNAprobe.

FISH Probe Reference V: Standard sales vial of HER2 PharmDx probe mix(K5331, Dako) containing blocking PNA. Overnight hybridization for 20hours.

All compositions were present as a single phase. The FISH probes wereincubated at 82° C. for 5 min and then at 45° C. for 60 minutes with noblocking, except for FISH Probe Reference V, which had PNA blocking andwas hybridized for 20 hours.

Results:

Signal Strength DNA Probes PNA Probes Composition Ia 0  ½ Composition Ib0  ½ Composition Ic ½ 2½ Composition IIa ½ 3 Composition IIb 1 2Composition IIc ½ 3 Composition IIIa 1 2½ Composition IIIb 1½  2½Composition IIIc 2 3 Composition IVa 2½-3 3 Composition IVb 3 3Composition IVc 3 3 Reference V 2 2½ NOTE: Composition IVa gave strongDNA signals with no salt. This is not possible with standard FISHcompositions, where DNA binding is salt dependent.

Example 10

This example compares the signal intensity from samples treated with thecompositions of the invention as a function of polar aprotic solvent anddextran sulfate concentration under high salt (4× normal) conditions.

FISH Probe Composition I: 0% ethylene carbonate, 29% dextran sulfate,1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL Texas Red labeled HER2gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Compositionwas a single phase.

FISH Probe Composition II: 5% ethylene carbonate, 27% dextran sulfate,1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL Texas Red labeled HER2gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Compositionwas a single phase.

FISH Probe Composition III: 10% ethylene carbonate, 25% dextran sulfate,1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL Texas Red labeled HER2gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Compositionwas a single phase.

FISH Probe Composition IV (not tested): 20% ethylene carbonate, 21%dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL TexasRed labeled HER2 gene DNA probe and 50 nM of FITC-labeled CEN-7 PNAprobe. Composition had two phases.

Results:

Signal Strength DNA Probes PNA Probes Composition I ½ 3 Composition II 22½ Composition III 3 3 Composition IV — — Note: Composition II gave goodDNA signals with only 5% EC and strong DNA signals with 10% EC.

Example 11

This example compares the signal intensity and background from samplestreated with different phases of the compositions of the invention.

FISH Probe Composition: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphatebuffer, 40% Ethylene carbonate, 8 ng/μL Texas Red labeled HER2 gene DNAprobe and 600 nM FITC-labeled CEN-17 PNA probe. The FISH probes wereincubated at 82° C. for 5 min and then at 45° C. for 60 minutes. Noblocking.

Results:

Signal Intensity DNA Probe PNA Probe Background Upper Phase 3 1½ +2Lower Phase 3 2½ +1 Mix of Upper and 2½ 3 +½ Lower Phases NOTE: theupper phase had more background than the lower phase in theseexperiments.

Example 12

This example is similar to the previous example, but uses a differentDNA probe and GBL instead of EC.

FISH Probe Composition: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphatebuffer, 40% GBL, 10 ng/μL Texas Red labeled CCND1 gene DNA probe and 600nM FITC-labeled CEN-17 PNA probe.

The FISH probes were incubated at 82° C. for 5 min and then at 45° C.for 60 minutes. No blocking.

Results:

Signal Strength DNA Probe PNA Probe Background Top Phase 3 0-½ +1½Bottom Phase 2 ½ +3 Mixed Phases 2½ ½ +2½

Example 13

This example examines the number of phases in the compositions of theinvention as a function of polar aprotic solvent and dextran sulfateconcentration.

FISH Probe Compositions: 10 or 20% dextran sulfate; 300 mM NaCl; 5 mMphosphate buffer; 0, 5, 10, 15, 20, 25, 30% EC; 10 ng/μL probe.

Results:

Number of Phases Number of Phases % EC 10% Dextran 20% Dextran 0 1 1 5 11 10 1 1 15 1 1 20 2 2 25 2 2 30 2 2 NOTE: 15% EC, 20% dextran sulfateproduces the nicest high signal intensities of the above one phasesolution. Two phases 20% EC has even higher signal intensities than 15%.(Data not shown).

Example 14

This example compares the signal intensity and background from samplestreated with different compositions of the invention as a function ofprobe concentration and hybridization time.

FISH Probe Composition I: 10 ng/μL HER2 TxRed labeled DNA probe(standard concentration) and standard concentration of CEN7 FITC labeledPNA probe (50 nM); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer.

FISH Probe Composition II: 5 ng/μL HER2 TxRed labeled DNA probe (½ ofstandard concentration) and standard concentration (50 nM) of FITClabeled CEN7 PNA probes; 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer.

FISH Probe Composition III: 2.5 ng/μL HER2 TxRed labeled DNA probe (¼ ofstandard concentration) and ½ of the standard concentration (25 nM) ofCEN7 PNA probes; 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer.

Compositions I-III existed as a single phase. The FISH probes wereincubated at 82° C. for 5 min and then at 45° C. for 3 hours, 2 hoursand 1 hours.

Results:

Hybrid- Signal Intensity ization I II III time DNA PNA B.G. DNA PNA B.G.DNA PNA B.G. 3 hours 3 3 +3 3 3 +2.5 3 3 +1.5 2 hours 2.5 2.5 +3 3 3 +33 3 +1.5 1 hours 2.5 2.5 +3 3 3 +1.5 2.5 3 +1 Signals scored as “3” wereclearly visible in a 20x objective. B.G.: Back ground.

Example 15

This example compares the signal intensity and background from samplestreated with the compositions of the invention as a function of blockingagent.

FISH Probe Compositions: 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer; 2.5 ng/μL HER2 TxRed labeled DNA probe (¼ of standardconcentration) and ½ of the standard concentration (300 nM) FITC labeledCEN17 PNA probe. Samples were blocked with: (a) nothing; (b) 0.1 μg/μLCOT1 (15279-011, Invitrogen); (c) 0.3 μg/μL COT1; or (d) 0.1 μg/μL totalhuman DNA before hybridization using the compositions of the invention.

All samples were present as a single phase. The FISH probes wereincubated at 82° C. for 5 min and then at 45° C. for 60 minutes.

Results:

Signal Intensity Blocking Agent Background DNA PNA Nothing +1-1.5 3 2.50.1 μg/μL COT1 +1 3 2.5 0.3 μg/μL COT1 +1.5 3 2.5 0.1 μg/μL total humanDNA +½ 3 2.5 NOTE: Background levels without blocking are significantlylower than what is normally observed by standard FISH with no blocking.In contrast, if a standard FISH composition does not contain a blockingagent, signals normally cannot be read.

Example 16

This experiment compares different ways of removing background stainingusing the compositions of the invention.

All compositions contained 15% EC, 20% dextran sulfate, 600 mM NaCl, 10mM phosphate buffer, 2.5 ng/μL HER2 DNA probes (¼ of standardconcentration), 300 nM CEN17 PNA probe (½ of standard concentration),and one of the following background-reducing agents:

A) 5 μM blocking-PNA (see Kirsten Yang Nielsen et al., PNA SuppressionMethod Combined with Fluorescence In Situ Hybridisation (FISH) TechniqueinPRINS and PNA Technologies in Chromosomal Investigation, Chapter 10(Franck Pellestor ed.) (Nova Science Publishers, Inc. 2006))B) 0.1 μg/μL COT-1 DNAC) 0.1 μg/μL total human DNA (THD) (sonicated unlabelled THD)D) 0.1 μg/μL sheared salmon sperm DNA (AM9680, Ambion)E) 0.1 μg/μL calf thymus DNA (D8661, Sigma)F) 0.1 μg/μL herring sperm DNA (D7290, Sigma)G) 0.5% formamideH) 2% formamideI) 1% ethylene glycol (1.09621, Merck)J) 1% glycerol (1.04095, Merck)K) 1% 1,3-Propanediol (533734, Aldrich)L) 1% H₂0 (control)

All samples were present as a single phase. The probes were incubated at82° C. for 5 minutes and then at 45° C. on FFPE tissue sections for 60and 120 minutes.

Results:

Signal Intensity Background blocking Hybridization/min Background DNAPNA Blocking-PNA 60 +1 3 2.5 Blocking-PNA 120 +1-1½ 3 2.5 COT-1 60 +½ 32.5 COT-1 120 +0-½  3 2.5 THD 60 +0 3 3 THD 120 +½ 3 2.5 Salmon DNAsperm 60 +0 3 3 Salmon DNA sperm 120 +0 3 3 Calf Thymus DNA 60 +0 2.5 3Calf Thymus DNA 120 +½ 3 2.5 Hearing sperm DNA 60 +0 3 3 Hearing spermDNA 120 +½ 2.5 3 0.5% formamide 60 +0 2.5 3 0.5% formamide 120 +0 3 3 2%formamide 60 +½ 2.5 3 2% formamide 120 +½ 3 3 1% Ethylene Glycol 60 +½2.5 3 1% Ethylene Glycol 120 +1½  3 2.5 1% Glycerol 60 +½ 0.5 3 1%Glycerol 120 +1 3 2.5 1% 1,3-Propanediol 60 +0 3 2.5 1% 1,3-Propanediol120 +1 3 2.5 Nothing 60 +1 2.5 2.5 Nothing 120 +1½  3 2.5 NOTE: allbackground reducing reagents, except for blocking-PNA, showed an effectin background reduction. Thus, specific blocking against repetitive DNAsequences is not required.

Example 17

This experiment compares the signal intensity from the upper and lowerphases using two different polar aprotic solvents.

FISH Probe Composition I: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% ethylene trithiocarbonate (ET) (E27750, Aldrich),5 μM blocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

FISH Probe Composition II: 10% dextran sulfate, 300 mM NaCl, 5 mMphosphate buffer, 40% glycol sulfite (GS) (G7208, Aldrich), 5 μMblocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.

The FISH probes were incubated at 82° C. for 5 min and then at 45° C.for 60 minutes.

Results:

Signal Intensity I (ET) II (GS) Upper Phase 1½ 0 Lower Phase 0 3 Mix ofUpper and Lower Phases 2½ 3

Example 18

This experiment examines the ability of various polar aprotic solventsto form a one-phase system.

All compositions contained: 20% dextran sulfate, 600 mM NaCl, 10 mMphosphate buffer, and either 10, 15, 20, or 25% of one of the followingpolar aprotic solvents:

Sulfolane

γ-Butyrolactone

Ethylene trithiocarbonate

Glycol sulfite

Propylene carbonate

Results: all of the polar aprotic solvents at all of the concentrationsexamined produced at least a two-phase system in the compositions used.However, this does not exclude that these compounds can produce aone-phase system under other composition conditions.

Example 19

This experiment examines the use of the compositions of the invention inchromogenic in situ hybridization (CISH) analysis on multi FFPE tissuesections.

FISH Probe Composition I: 4.5 ng/μL TCRAD FITC labelled gene DNA probe(¼ of standard concentration) (RP11-654A2, RP11-246A2, CTP-2355L21,RP11-158G6, RP11-780M2, RP11-481C14; size 1018 kb); 15% EC; 20% dextransulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.

FISH Probe Composition II: 4.5 ng/μL TCRAD FITC labelled gene DNA probe(¼ of standard concentration) (size 1018 kb); 15% EC; 20% dextransulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL shearedsalmon DNA sperm.

FISH Probe Composition III: 300 nM of each individual FITC labelled PNACEN17 probe (½ of standard concentration); 15% EC; 20% dextran sulfate;600 mM NaCl; 10 mM citrate buffer, pH 6.0.

All samples were analyzed using the Dako DuoCISH protocol (SK108) andcompositions for split probes with the exception that the stringencywash was conducted for 20 minutes instead of 10 minutes, and withoutusing the DuoCISH red chromogen step.

Results:

Signal Strength Composition FITC DNA FITC PNA I 3 — II 3 — III — 3 Note:The signal intensities were very strong. Due to the high levels ofbackground, it was not possible to discriminate if addition of salmonsperm DNA in Composition II reduced the background. Signals were clearlyvisible using a 10x objective in e.g. tonsils, which in general had lessbackground. If tissues possessed high background, the signals wereclearly visible using a 20x objective.

Example 20

This example compares the signal intensity and background from FFPEtissue sections treated with the compositions of the invention with twoDNA probes.

FISH Probe Composition I: 9 ng/μL IGH FITC labelled gene DNA probe(RP11-151B17, RP11-112H5, RP11-101G24, RP11-12F16, RP11-47P23,CTP-3087C18; size 612 kb); 6.4 ng/μL MYC Tx Red labeled DNA probe(CTD-2106F24, CTD-2151C21, CTD-2267H22; size 418 kb); 15% EC; 20%dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.

FISH Probe Composition II: 9 ng/μL IGH FITC labelled gene DNA probe; 6.4ng MYC TxRed labeled DNA probe; 15% EC, 20% dextran sulfate; 600 mMNaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL sheared salmon sperm DNA.

Signal Strength Salmon DNA FITC probe Texas Red probe Background − 2½ 2½+2.5 + 3 3 +1.5 NOTE: the high background was probably due to the factthat standard probe concentrations were used.

Example 21

This experiment examines the use of the compositions of the invention oncytological samples.

FISH Probe Composition: 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mMphosphate buffer; 5 ng/μL HER2 TxRed labeled DNA probe (½ of standardconcentration) and ½ of the standard concentration of CEN7 (25 nM).

The FISH probes were incubated on metaphase chromosome spreads at 82° C.for 5 minutes, then at 45° C. for 30 minutes, all without blocking.

Results:

Signal Strength DNA Probe PNA Probe Background 3 3 +1 No chromosomebanding (R-banding pattern) was observed with the compositions of theinvention, in contrast with traditional ISH solutions, which typicallyshow R-banding. A low homogenously red background staining of theinterphase nuclei and metaphase chromosomes was observed.

Example 22

This example compares the signal intensity and background from DNAprobes on cytology samples, metaphase spreads, with and withoutblocking.

FISH Probe Composition I: 6 ng/μL TCRAD Texas Red labelled gene DNAprobe (standard concentration) (CTP-31666K20, CTP-2373N7; size 301 kb)and 4.5 ng/μL FITC labelled gene DNA probe (¼ of standardconcentration); 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citratebuffer, pH 6.0.

FISH Probe Composition II: 6 ng/μL TCRAD Texas Red labelled gene DNAprobe (standard concentration) (size 301 kb) and 4.5 ng/μL FITC labelledgene DNA probe (¼ of standard concentration); 15% EC, 20% dextransulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL shearedsalmon sperm DNA.

The FISH probes were incubated on metaphase spreads at 82° C. for 5 min,then at 45° C. for 60 min.

Results:

Signal Intensity Blocking Agent Background Tx Red FITC Nothing +0 3 30.1 μg/μL Salmon DNA +0 3 3 Again, no chromosome banding (R-bandingpattern) was observed with the compositions of the invention. Inaddition, no background staining of the interphase nuclei or themetaphase chromosomes were observed.

Example 23

This example compares the staining intensity from mRNA in samplestreated with the compositions of the invention or formamidehybridization solutions as a function of hybridization time andtemperature.

FISH Probe Composition I: 20 nM fluorescein-labelled kappa PNA probe(Y5202, Dako); 30% formamide; 10% dextran sulfate; 10 mM NaCl; 50 mMTris, 0.1% sodium pyrophosphate, 0.2% polyvinylpyrrolidon, 0.2% Ficoll,5 mM Na₂EDTA

FISH Probe Composition II: 20 nM fluorescein-labelled kappa PNA probe(Y5202, Dako), 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citratebuffer, pH 6.0.

FISH Probe Composition III: 20 nM fluorescein-labelled kappa LNA probe,15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.

The stringent wash was performed at 55° C. for the PNA probe (FISH ProbeCompositions I and II) and at 30° C. for the LNA probe (FISH ProbeComposition III) for 25 min.

Results:

Hybridization Staining Intensity Probe time/temp Tonsil Colon PNA I  5min/55° C. 3 2 PNA I 15 min/55° C. 3 — PNA I 30 min/55° C. — 2 PNA I 60min/55° C. — 2½ PNA I 90 min/55° C. — 2½ PNA II  5 min/55° C. 3 2½-3 PNAII 15 min/55° C.  2-3   2-2½ PNA II 30 min/55° C. 3 — PNA II 60 min/55°C.   2-2½   2-2½ PNA II 90 min/55° C.   2-2½   2-2½ LNA III  5 min/42°C. 1½-2 1½ LNA III 15 min/42° C. 2½ 2½ LNA III 30 min/42° C. 2½-3 2½-3LNA III 60 min/42° C. 2½-3 2½-3 LNA III 90 min/42° C. 3 3 *— indicatesthat the tissue was over-digested and could not be read properly.

Negative controls having a Fluorescein-conjugated random PNA probe inFISH Probe Composition I and negative controls having no probe in FISHProbe Compositions II and III gave no signals (data not shown).

A positive control having a fluorescein-conjugated PNA probe directedagainst glyceraldehyde 3-phosphate dehydrogenase in FISH ProbeComposition I provided strong signals (data not shown).

No staining were observed for tissue types other than colon and tonsilon the small multi tissue section (data not shown).

These results show that RNA can be detected using the new buffer withboth LNA and PNA based probes. These results also show that signals forPNA in FISH Probe Compositions II and III were stronger than for PNA inFISH Probe Composition I at 5 min. Thus, the buffers of the inventioncan be used in place of traditional formamide buffers for affecting thestringency conditions of both natural and artificial bases such as e.g.DNA, RNA, PNA and LNA.

Example 24

This example compares staining intensity of PNA and LNA probeshybridized to mRNA in biological samples exposed to the same digestionincubation time.

FISH Probe Composition I: Fluorescein-conjugated random PNA probes, 30%formamide; 10% dextran sulfate; 10 mM NaCl; 50 mM Tris, 0.1% sodiumpyrophosphate, 0.2% polyvinylpyrrolidon, 0.2% Ficoll, 5 mM Na₂EDTA.

FISH Probe Composition II: 20 nM fluorescein-labelled kappa PNA probe(Flu-Flu-CTGCTGAGGCTGTAG, Panagen, Korea), 15% EC, 20% dextran sulfate;600 mM NaCl; 10 mM citrate buffer, pH 6.0.

FISH Probe Composition III: 20 nM fluorescein-labelled kappa LNA probe(Flu-CTGCTGAGGCTGTAG-Flu, Exicon, Denmark), 15% EC, 20% dextran sulfate;600 mM NaCl; 10 mM citrate buffer, pH 6.0.

The digestion time for all samples was 5 min.

Results:

Hybridization Staining Intensity Probe time/temp Tonsil Colon Control I90 min/35° C. 0 0 EC PNA II 90 min/45° C. 3 3 EC LNA III 90 min/35° C. 31½ No staining was observed for kidney and mamacarcinoma tissues. Thesamples showed low background, a nice morphology, and were notoverdigested.

These results further illustrate that the buffers of the invention canbe used for RNA hybridizations using both PNA and LNA probes.

Example 25

This example compares staining intensity of PNA and LNA probeshybridized to mRNA in biological samples exposed to the same digestionincubation time and hybridization temperature.

FISH Probe Composition I: Fluorescein-conjugated random PNA probes, 30%formamide (FM); 10% dextran sulfate; 10 mM NaCl; 50 mM Tris, 0.1% sodiumpyrophosphate, 0.2% polyvinylpyrrolidon, 0.2% Ficoll, 5 mM Na₂EDTA.

FISH Probe Composition II: 15% GBL, 20% dextran sulfate; 600 mM NaCl; 10mM citrate buffer, pH 6.2.

FISH Probe Composition III: 15% SL, 20% dextran sulfate; 600 mM NaCl; 10mM citrate buffer, pH 6.2.

FISH Probe Composition IV: 20 nM fluorescein-labelled kappa PNA probe(Flu-Flu-CTGCTGAGGCTGTAG, Panagen, Korea), 15% GBL, 20% dextran sulfate;600 mM NaCl; 10 mM citrate buffer, pH 6.2.

FISH Probe Composition V: 40 nM fluorescein-labelled kappa LNA probe(Flu-CTGCTGAGGCTGTAG-Flu, Exicon, Denmark), 15% GBL, 20% dextransulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.2.

FISH Probe Composition VI: 20 nM fluorescein-labelled kappa PNA probe(Flu-Flu-CTGCTGAGGCTGTAG, Panagen, Korea), 15% SL, 20% dextran sulfate;600 mM NaCl; 10 mM citrate buffer, pH 6.2.

FISH Probe Composition VII: 40 nM fluorescein-labelled kappa LNA probe(Flu-CTGCTGAGGCTGTAG-Flu, Exicon, Denmark), 15% SL, 20% dextran sulfate;600 mM NaCl; 10 mM citrate buffer, pH 6.2.

The digestion time for all samples was 5 min. The samples werehybridized at 45° C. for 90 min and stringent washed at 45° C. for 25min.

Results:

Staining Intensity Probe Background Tonsil Colon Mamma Kidney FM PNA I+0 2 ½ 0 0 GBL control II +0 0 0 0 0 SL Control III +0 0 0 0 0 GBL PNAIV +0 2½ 1½  0 0 GBL LNA V +0 2 ½ 0 0 SL PNA VI +0 2-2½ ½ 0 0 SL LNA VII+0 3 1½-2 0 0 No staining was observed for kidney and mamacarcinomatissues. The samples showed no background, nice morphology, and were notover-digested.

The results showed that signals for PNA in FISH Probe Compositions IVand VI were stronger than for PNA in FISH Probe Composition I (i.e.,traditional formamide buffer).

Further Embodiments Embodiment 1

A hybridization composition for RNA hybridization applicationscomprising at least one nucleic acid sequence, at least one polaraprotic solvent in an amount effective to enable hybridization to RNA,and a hybridization solution, wherein the polar aprotic solvent is notdimethyl sulfoxide (DMSO).

Embodiment 2

The hybridization composition according to embodiment 1, wherein theconcentration of polar aprotic solvent is about 1% to 95% (v/v)

Embodiment 3

The hybridization composition according to embodiment 1 or 2, whereinthe concentration of polar aprotic solvent is 5% to 10% (v/v).

Embodiment 4

The hybridization composition according to embodiment 1 or 2, whereinthe concentration of polar aprotic solvent is 10% to 20% (v/v).

Embodiment 5

The hybridization composition according to embodiment 1 or 2, whereinthe concentration of polar aprotic solvent is 20% to 30% (v/v).

Embodiment 6

The hybridization composition according to any one of embodiments 1 to5, wherein the polar aprotic solvent is non-toxic.

Embodiment 7

The hybridization composition according to any one of embodiments 1 to6, with the proviso that the composition does not contain formamide.

Embodiment 8

The hybridization composition according to embodiment 6, with theproviso that the composition contains less than 10% formamide.

Embodiment 9

The hybridization composition according to embodiment 8, with theproviso that the composition contains less than 2% formamide.

Embodiment 10

The hybridization composition according to embodiment 9, with theproviso that the composition contains less than 1% formamide.

Embodiment 11

The hybridization composition according to any of embodiments 1 to 10,wherein the polar aprotic solvent has lactone, sulfone, nitrile,sulfite, and/or carbonate functionality.

Embodiment 12

The hybridization composition according to any one of embodiments 1 to11, wherein the polar aprotic solvent has a dispersion solubilityparameter between 17.7 to 22.0 MPa^(1/2), a polar solubility parameterbetween 13 to 23 MPa^(1/2), and a hydrogen bonding solubility parameterbetween 3 to 13 MPa^(1/2).

Embodiment 13

The hybridization composition according to any one of embodiments 1 to12, wherein the polar aprotic solvent has a cyclic base structure.

Embodiment 14

The hybridization composition according to any one of embodiments 1 to13, wherein the polar aprotic solvent is selected from the groupconsisting of:

where X is O and R1 is alkyldiyl, and

where X is optional and if present, is chosen from O or S,where Z is optional and if present, is chosen from O or S,where A and B independently are O or N or S or part of the alkyldiyl ora primary amine,where R is alkyldiyl, andwhere Y is O or S or C.

Embodiment 15

The hybridization composition according to any one of embodiments 1 to14, wherein the polar aprotic solvent is selected from the groupconsisting of: acetanilide, acetonitrile, N-acetyl pyrrolidone, 4-aminopyridine, benzamide, benzimidazole, 1,2,3-benzotriazole,butadienedioxide, 2,3-butylene carbonate, γ-butyrolactone, caprolactone(epsilon), chloro maleic anhydride, 2-chlorocyclohexanone,chloroethylene carbonate, chloronitromethane, citraconic anhydride,crotonlactone, 5-cyano-2-thiouracil, cyclopropylnitrile, dimethylsulfate, dimethyl sulfone, 1,3-dimethyl-5-tetrazole, 1,5-dimethyltetrazole, 1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,epsilon-caprolactam, ethanesulfonylchloride, ethyl ethyl phosphinate,N-ethyl tetrazole, ethylene carbonate, ethylene trithiocarbonate,ethylene glycol sulfate, glycol sulfite, furfural, 2-furonitrile,2-imidazole, isatin, isoxazole, malononitrile, 4-methoxy benzonitrile,1-methoxy-2-nitrobenzene, methyl alpha bromo tetronate, 1-methylimidazole, N-methyl imidazole, 3-methyl isoxazole, N-methylmorpholine-N-oxide, methyl phenyl sulfone, N-methyl pyrrolidinone,methyl sulfolane, methyl-4-toluenesulfonate, 3-nitroaniline,nitrobenzimidazole, 2-nitrofuran, 1-nitroso-2-pyrrolidinone,2-nitrothiophene, 2-oxazolidinone, 9,10-phenanthrenequinone, N-phenylsydnone, phthalic anhydride, picolinonitrile (2-cyanopyridine),1,3-propane sultone, β-propiolactone, propylene carbonate,4H-pyran-4-thione, 4H-pyran-4-one (γ-pyrone), pyridazine, 2-pyrrolidone,saccharin, succinonitrile, sulfanilamide, sulfolane,2,2,6,6-tetrachlorocyclohexanone, tetrahydrothiapyran oxide,tetramethylene sulfone (sulfolane), thiazole, 2-thiouracil,3,3,3-trichloro propene, 1,1,2-trichloro propene, 1,2,3-trichloropropene, trimethylene sulfide-dioxide, and trimethylene sulfite.

Embodiment 16

The hybridization composition according to any one of embodiments 1 to14, wherein the polar aprotic solvent is selected from the groupconsisting of:

Embodiment 17

The hybridization composition according to any one of embodiments 1 to14, wherein the polar aprotic solvent is:

Embodiment 18

The hybridization composition according to any one of embodiments 1 to17, further comprising at least one additional component selected fromthe group consisting of: buffering agents, salts, accelerating agents,chelating agents, detergents, and blocking agents.

Embodiment 19

The hybridization composition according to embodiment 18, wherein theaccelerating agent is dextran sulfate and the salts are NaCl and/orphosphate buffer.

Embodiment 20

The hybridization composition according to embodiment 19, wherein thedextran sulfate is present at a concentration of 5% to 40%, the NaCl ispresent at a concentration of 0 mM to 1200 mM, and/or the phosphatebuffer is present at a concentration of 0 mM to 50 mM.

Embodiment 21

The hybridization composition according to embodiment 20, wherein thedextran sulfate is present at a concentration of 10% to 30%, the NaCl ispresent at a concentration of 300 mM to 600 mM, and/or the phosphatebuffer is present at a concentration of 5 mM to 20 mM.

Embodiment 22

The hybridization composition according to embodiment 18, wherein theaccelerating agent is selected from the group consisting of: formamide,DMSO, glycerol, propylene glycol, 1,2-propanediol, diethylene glycol,ethylene glycol, glycol, and 1,3 propanediol, and the buffering agent iscitric acid buffer.

Embodiment 23

The hybridization composition according to embodiment 22, wherein theformamide is present at a concentration of 0.1-5%, the DMSO is presentat a concentration of 0.01% to 10%, the glycerol, propylene glycol,1,2-propanediol, diethylene glycol, ethylene glycol, glycol, and 1,3propanediol are present at a concentration of 0.1% to 10%, and thecitric acid buffer is present at a concentration of 1 mM to 50 mM.

Embodiment 24

The hybridization composition according to embodiment 18, wherein theblocking agent is selected from the group consisting of: total humanDNA, herring sperm DNA, salmon sperm DNA, and calf thymus DNA.

Embodiment 25

The hybridization composition according to embodiment 24, wherein thetotal human DNA, herring sperm DNA, salmon sperm DNA, and calf thymusDNA are present at a concentration of 0.01 to 10 μg/μL.

Embodiment 26

The hybridization composition according to any one of embodiments 1-25,comprising 40% of at least one polar aprotic solvent, 10% dextransulfate, 300 mM NaCl, and 5 mM phosphate buffer.

Embodiment 27

The hybridization composition according to any one of embodiments 1-25,comprising 15% of at least one polar aprotic solvent, 20% dextransulfate, 600 mM NaCl, 10 mM phosphate buffer, and 0.1 μg/μl total humanDNA.

Embodiment 28

The hybridization composition according to any one of embodiments 1-25,comprising 15% of at least one polar aprotic solvent, 20% dextransulfate, 600 mM NaCl, 10 mM citric acid buffer pH 6.2, and 0.1 μg/μLherring sperm DNA, or salmon sperm DNA, or calf thymus DNA, or 0.5%formamide, or 1% ethylene glycol, or 1% 1,3 propanediol.

Embodiment 29

The hybridization composition according to any one of embodiments 1-28,comprising more than one phase at room temperature.

Embodiment 30

The hybridization composition according to embodiment 29, comprising twophases at room temperature.

Embodiment 31

The hybridization composition according to embodiment 29, comprisingthree phases at room temperature.

Embodiment 32

The hybridization composition according to any one of embodiments 1-31,wherein the RNA is messenger RNA (mRNA), viral RNA, small interferingRNA (siRNA), small nuclear RNA (snRNA), non-coding RNA (ncRNA, e.g.,tRNA and rRNA), transfer messenger RNA (tmRNA), piwi-interacting RNA(piRNA), long noncoding RNA, small nucleolar RNA (snoRNA), antisenseRNA, double-stranded RNA (dsRNA), or heterogeneous nuclear RNA (hnRNA).

Embodiment 33

The hybridization composition according to any one of embodiments 1-32,wherein the at least one nucleic acid sequence is a PNA sequence, an LNAsequence, or a DNA sequence.

Embodiment 34

A hybridization method comprising:

-   -   providing an RNA sequence,    -   providing a second nucleic acid sequence,    -   providing an aqueous composition comprising at least one polar        aprotic solvent in an amount effective to enable hybridization        to RNA, and    -   combining the RNA and the second nucleic acid sequence and the        aqueous composition for at least a time period sufficient to        hybridize the RNA and the second nucleic acid sequence,    -   wherein the polar aprotic solvent is not dimethyl sulfoxide        (DMSO).

Embodiment 35

A hybridization method comprising:

-   -   providing an RNA sequence, and    -   applying to said RNA sequence an aqueous composition comprising        a second nucleic acid sequence and at least one polar aprotic        solvent in an amount effective to enable hybridization to RNA        for at least a time period sufficient to hybridize the RNA and        the second nucleic acid sequences,    -   wherein the polar aprotic solvent is not dimethyl sulfoxide        (DMSO).

Embodiment 36

A hybridization method comprising:

-   -   providing an RNA sequence, and    -   applying a hybridization composition according to any of        embodiments 1-31 to said RNA sequence for at least a time period        sufficient to hybridize the RNA and the second nucleic acid        sequences.

Embodiment 37

The method according to embodiments 34 or 35, wherein the polar aproticsolvent is defined according to any of embodiments 2-6 or 11-17.

Embodiment 38

The method according to any of embodiments 34-37, wherein a sufficientamount of energy to hybridize the RNA and the second nucleic acids isprovided.

Embodiment 39

The method according to embodiment 38, wherein the energy is provided byheating the hybridization composition and nucleic acid sequence.

Embodiment 40

The method according to embodiment 39, wherein the heating step isperformed by the use of microwaves, hot baths, hot plates, heat wire,peltier element, induction heating or heat lamps.

Embodiment 41

The method according to any one of embodiments 34-40, further comprisinga denaturation step.

Embodiment 42

The method according to embodiment 41, wherein the denaturation andhybridization steps occur separately.

Embodiment 43

The method according to any one of embodiments 34-42, wherein the stepof hybridizing includes the steps of heating and cooling thehybridization composition and nucleic acid sequences.

Embodiment 44

The method according to any one of embodiments 34-43, wherein thehybridization step takes less than 8 hours.

Embodiment 45

The method according to embodiment 44, wherein the hybridization steptakes less than 1 hour.

Embodiment 46

The method according to embodiment 45, wherein the hybridization steptakes less than 30 minutes.

Embodiment 47

The method according to embodiment 46, wherein the hybridization steptakes less than 15 minutes.

Embodiment 48

The method according to embodiment 47, wherein the hybridization steptakes less than 5 minutes.

Embodiment 49

The method according to any one of embodiments 34-48, wherein the secondnucleic acid is a PNA sequence, an LNA sequence, or a DNA sequence.

Embodiment 50

The method according to any one of embodiments 34-49, wherein the RNA ismessenger RNA (mRNA), viral RNA, small interfering RNA (siRNA), smallnuclear RNA (snRNA), non-coding RNA (ncRNA, e.g., tRNA and rRNA),transfer messenger RNA (tmRNA), micro RNA (miRNA), piwi-interacting RNA(piRNA), long noncoding RNA, small nucleolar RNA (snoRNA), antisenseRNA, double-stranded RNA (dsRNA), and heterogeneous nuclear RNA (hnRNA).

Embodiment 51

The method according to any one of embodiments 34-50, wherein the RNA isin a biological sample.

Embodiment 52

The method according to embodiment 51, wherein the biological sample isa cytology or histology sample.

Embodiment 53

The method according to any one of embodiments 34-52, wherein thehybridization composition comprises one phase at room temperature.

Embodiment 54

The method according to any one of embodiments 34-52, wherein thehybridization composition comprises multiple phases at room temperature.

Embodiment 55

The method according to embodiment 54, wherein the hybridizationcomposition comprises two phases at room temperature.

Embodiment 56

The method according to embodiment 54 or 55, wherein the phases of thehybridization composition are mixed.

Embodiment 57

The method according to any one of embodiments 34-56, further comprisinga blocking step.

Embodiment 58

Use of a hybridization composition comprising between 1 and 95% (v/v) ofat least one polar aprotic solvent in an RNA hybridization application.

Embodiment 59

Use of a composition according to embodiment 58, wherein thehybridization composition is according to any one of embodiments 1 to33.

The invention claimed is:
 1. A composition comprising at least one polaraprotic solvent in an amount effective to enable hybridization of anucleic acid sequence to an RNA sequence within a cell in a samplehaving a preserved cell morphology, and at least 10% dextran sulfate,wherein the polar aprotic solvent is not dimethyl sulfoxide (DMSO). 2.The composition according to claim 1, wherein the concentration of polaraprotic solvent is about 1% to 90% (v/v).
 3. The composition accordingto claim 2, wherein the concentration of polar aprotic solvent is 5% to10% (v/v).
 4. The composition according to claim 2, wherein theconcentration of polar aprotic solvent is 10% to 20% (v/v).
 5. Thecomposition according to claim 2, wherein the concentration of polaraprotic solvent is 20% to 30% (v/v).
 6. The composition according toclaim 1, wherein the polar aprotic solvent is non-toxic.
 7. Thecomposition according to claim 1, with the proviso that the compositiondoes not contain formamide.
 8. The composition according to claim 6,with the proviso that the composition contains less than 10% formamide.9. The composition according to claim 8, with the proviso that thecomposition contains less than 2% formamide.
 10. The compositionaccording to claim 9, with the proviso that the composition containsless than 1% formamide.
 11. The composition according to claim 1,wherein the polar aprotic solvent has lactone, sulfone, nitrile,sulfite, and/or carbonate functionality.
 12. The composition accordingto claim 1, wherein the polar aprotic solvent has a dispersionsolubility parameter between 17.7 to 22.0 MPa^(1/2), a polar solubilityparameter between 13 to 23 MPa^(1/2), and a hydrogen bonding solubilityparameter between 3 to 13 MPa^(1/2).
 13. The composition according toclaim 1, wherein the polar aprotic solvent has a cyclic base structure.14. The composition according to claim 1, wherein the polar aproticsolvent is selected from the group consisting of:

where X is O and R1 is alkyldiyl, and

where X is optional and if present, is chosen from O or S, where Z isoptional and if present, is chosen from O or S, where A and Bindependently are O or N or S or part of the alkyldiyl or a primaryamine, where R is alkyldiyl, and where Y is O or S or C, and wherein ifY is C, then either X or Z is not present or both X and Z are notpresent.
 15. The composition according to claim 1, wherein the polaraprotic solvent is selected from the group consisting of: acetanilide,acetonitrile, N-acetyl pyrrolidone, 4-amino pyridine, benzamide,benzimidazole, 1,2,3-benzotriazole, butadienedioxide, 2,3-butylenecarbonate, γ-butyrolactone, caprolactone (epsilon), chloro maleicanhydride, 2-chlorocyclohexanone, chloroethylene carbonate,chloronitromethane, citraconic anhydride, crotonlactone,5-cyano-2-thiouracil, cyclopropylnitrile, dimethyl sulfate, dimethylsulfone, 1,3-dimethyl-5-tetrazole, 1,5-dimethyl tetrazole,1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,epsilon-caprolactam, ethanesulfonylchloride, ethyl ethyl phosphinate,N-ethyl tetrazole, ethylene carbonate, ethylene trithiocarbonate,ethylene glycol sulfate, glycol sulfite, furfural, 2-furonitrile,2-imidazole, isatin, isoxazole, malononitrile, 4-methoxy benzonitrile,1-methoxy-2-nitrobenzene, methyl alpha bromo tetronate, 1-methylimidazole, N-methyl imidazole, 3-methyl isoxazole, N-methylmorpholine-N-oxide, methyl phenyl sulfone, N-methyl pyrrolidinone,methyl sulfolane, methyl-4-toluenesulfonate, 3-nitroaniline,nitrobenzimidazole, 2-nitrofuran, 1-nitroso-2-pyrrolidinone,2-nitrothiophene, 2-oxazolidinone, 9,10-phenanthrenequinone, N-phenylsydnone, phthalic anhydride, picolinonitrile (2-cyanopyridine),1,3-propane sultone, β-propiolactone, propylene carbonate,4H-pyran-4-thione, 4H-pyran-4-one (γ-pyrone), pyridazine, 2-pyrrolidone,saccharin, succinonitrile, sulfanilamide, sulfolane,2,2,6,6-tetrachlorocyclohexanone, tetrahydrothiapyran oxide,tetramethylene sulfone (sulfolane), thiazole, 2-thiouracil,3,3,3-trichloro propene, 1,1,2-trichloro propene, 1,2,3-trichloropropene, trimethylene sulfide-dioxide, and trimethylene sulfite.
 16. Thecomposition according to claim 1, wherein the polar aprotic solvent isselected from the group consisting of:


17. The composition according to claim 1, wherein the polar aproticsolvent is:


18. The composition according to claim 1, further comprising at leastone additional component selected from the group consisting of:buffering agents, salts, accelerating agents, chelating agents,detergents, and blocking agents.
 19. The composition according to claim18, wherein the salt is NaCl and/or the buffering agent is phosphatebuffer.
 20. The composition according to claim 19, wherein the dextransulfate is present at a concentration of 10% to 40%, the NaCl is presentat a concentration of 0 mM to 1200 mM, and/or the phosphate buffer ispresent at a concentration of 0 mM to 50 mM.
 21. The compositionaccording to claim 20, wherein the dextran sulfate is present at aconcentration of 10% to 30%, the NaCl is present at a concentration of300 mM to 600 mM, and/or the phosphate buffer is present at aconcentration of 5 mM to 20 mM.
 22. The composition according to claim18, wherein the accelerating agent is selected from the group consistingof: formamide, DMSO, glycerol, propylene glycol, 1,2-propanediol,diethylene glycol, ethylene glycol, glycol, and 1,3 propanediol, and thebuffering agent is citric acid buffer.
 23. The composition according toclaim 22, wherein the formamide is present at a concentration of 0.1-5%,the DMSO is present at a concentration of 0.01% to 10%, the glycerol,propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol,glycol, and 1,3 propanediol are present at a concentration of 0.1% to10%, and the citric acid buffer is present at a concentration of 1 mM to50 mM.
 24. The composition according to claim 18, wherein the blockingagent is selected from the group consisting of: total human DNA, herringsperm DNA, salmon sperm DNA, and calf thymus DNA.
 25. The compositionaccording to claim 24, wherein the total human DNA, herring sperm DNA,salmon sperm DNA, and calf thymus DNA are present at a concentration of0.01 to 10 μg/μL.
 26. The composition according to claim 1, comprising40% of at least one polar aprotic solvent, 10% dextran sulfate, 300 mMNaCl, and 5 mM phosphate buffer.
 27. The composition according to claim1, comprising 15% of at least one polar aprotic solvent, 20% dextransulfate, 600 mM NaCl, 10 mM phosphate buffer, and 0.1 μg/μl total humanDNA.
 28. The composition according to claim 1, comprising 15% of atleast one polar aprotic solvent, 20% dextran sulfate, 600 mM NaCl, 10 mMcitric acid buffer pH 6.2, and 0.1 μg/μL herring sperm DNA, or salmonsperm DNA, or calf thymus DNA, or 0.5% formamide, or 1% ethylene glycol,or 1% 1,3 propanediol.
 29. The composition according to claim 1,comprising more than one phase at room temperature.
 30. The compositionaccording to claim 29, comprising two phases at room temperature. 31.The composition according to claim 29, comprising three phases at roomtemperature.
 32. The composition according to claim 1, wherein the RNAwithin the sample having a preserved cell morphology is messenger RNA(mRNA), viral RNA, small interfering RNA (siRNA), small nuclear RNA(snRNA), non-coding RNA (ncRNA), transfer RNA (tRNA), ribosomal RNA(rRNA), transfer messenger RNA (tmRNA), piwi-interacting RNA (piRNA),long noncoding RNA, small nucleolar RNA (snoRNA), antisense RNA,double-stranded RNA (dsRNA), or heterogeneous nuclear RNA (hnRNA).
 33. Ahybridization method comprising: combining a sample having a preservedcell morphology comprising an RNA sequence, a nucleic acid sequence, andan aqueous composition for at least a time period sufficient tohybridize the RNA sequence within the cell with the nucleic acidsequence, wherein the aqueous composition comprises at least one polaraprotic solvent in an amount effective to enable hybridization of thenucleic acid sequence to an RNA sequence within a cell and the samplemorphology is preserved, and at least 10% dextran sulfate, wherein thepolar aprotic solvent is not dimethyl sulfoxide (DMSO).
 34. Ahybridization method comprising: combining a nucleic acid sequence withthe composition according to claim 1; applying the combination of thenucleic acid sequence and the composition according to claim 1 to asample having a preserved cell morphology Comprising an RNA sequence forat least a time period sufficient to hybridize the RNA with the nucleicacid sequences within the cell and the sample morphology is preserved.35. The method according to claim 34, wherein a sufficient amount ofenergy to hybridize the RNA within the cell and the nucleic acidsequences is added.
 36. The method according to claim 35, wherein theenergy is provided by heating the composition, the cell comprising theRNA, and the nucleic acid sequence.
 37. The method according to claim36, wherein the heating step is performed by the use of one or moremicrowaves, hot baths, hot plates, heat wires, peltier elements,induction heating or heat lamps.
 38. The method according to claim 34,wherein the nucleic acid is double stranded and further comprising adenaturation step.
 39. The method according to claim 38, wherein thedenaturation and hybridization steps occur separately.
 40. The methodaccording to claim 34, wherein the step of hybridizing includes thesteps of heating and cooling the composition, the cell comprising theRNA, and the nucleic acid sequence.
 41. The method according to claim34, wherein the hybridization step takes less than 8 hours.
 42. Themethod according to claim 41, wherein the hybridization step takes lessthan 1 hour.
 43. The method according to claim 42, wherein thehybridization step takes less than 30 minutes.
 44. The method accordingto claim 43, wherein the hybridization step takes less than 15 minutes.45. The method according to claim 44, wherein the hybridization steptakes less than 5 minutes.
 46. The method according to claim 34, whereinthe cell comprising the RNA sequence is in a biological sample.
 47. Themethod according to claim 46, wherein the biological sample is acytology or histology sample.
 48. The method according to claim 34,wherein the composition comprises one phase at room temperature.
 49. Themethod according to claim 34, wherein the composition comprises multiplephases at room temperature.
 50. The method according to claim 49,wherein the composition comprises two phases at room temperature. 51.The method according to claim 49, wherein the phases of the compositionare mixed.
 52. The method according to claim 34, further comprising ablocking step.
 53. The composition according to claim 1, furthercomprising at least one nucleic acid sequence.
 54. The compositionaccording to claim 53, wherein the at least one nucleic acid sequence isa PNA sequence, an LNA sequence, or a DNA sequence.